U.S. patent application number 11/311886 was filed with the patent office on 2007-11-29 for nucleic acid molecules and other molecules associated with plants.
Invention is credited to Joseph R. Byrum.
Application Number | 20070277267 11/311886 |
Document ID | / |
Family ID | 38750995 |
Filed Date | 2007-11-29 |
United States Patent
Application |
20070277267 |
Kind Code |
A1 |
Byrum; Joseph R. |
November 29, 2007 |
Nucleic acid molecules and other molecules associated with
plants
Abstract
The present invention is in the field of plant genetics. More
specifically the invention relates to nucleic acid molecules and
nucleic acid molecules that contain markers, in particular, single
nucleotide polymorphism (SNP) and repetitive element markers. In
addition, the present invention provides nucleic acid molecules
having regulatory elements or encoding proteins or fragments
thereof. The invention also relates to proteins and fragments of
proteins so encoded and antibodies capable of binding the proteins.
The invention also relates to methods of using the nucleic acid
molecules, markers, repetitive elements and fragments of repetitive
elements, regulatory elements, proteins and fragments of
proteins.
Inventors: |
Byrum; Joseph R.; (Des
Moines, IA) |
Correspondence
Address: |
ARNOLD & PORTER, LLP
555 TWELFTH STREET, N.W.
ATTN: IP DOCKETING
WASHINGTON
DC
20004
US
|
Family ID: |
38750995 |
Appl. No.: |
11/311886 |
Filed: |
December 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09421106 |
Oct 15, 1999 |
|
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11311886 |
Dec 20, 2005 |
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Current U.S.
Class: |
800/285 ;
530/370; 536/23.6 |
Current CPC
Class: |
C12Q 1/6895 20130101;
C12N 15/8242 20130101; C12Q 2600/156 20130101; Y02A 40/146
20180101; C07K 14/415 20130101; C12N 15/8261 20130101 |
Class at
Publication: |
800/285 ;
530/370; 536/023.6 |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07K 14/415 20060101 C07K014/415; C07H 21/02 20060101
C07H021/02 |
Claims
1. A substantially purified nucleic acid molecule, said nucleic
acid molecule capable of specifically hybridizing to a second
nucleic acid molecule having the nucleic acid sequence of SEQ ID
NO: 1 or complement or fragment thereof.
2. The substantially purified nucleic acid molecule according to
claim 1, wherein said nucleic acid molecule comprises a
microsatellite sequence.
3. The substantially purified nucleic acid molecule according to
claim 1, wherein said nucleic acid molecule comprises a region
having a single nucleotide polymorphism.
4. The substantially purified nucleic acid molecule according to
claim 1, wherein said nucleic acid molecule comprises a nucleic
acid molecule having the nucleic acid sequence of SEQ ID NO: 1 or
the complement thereof.
5. The substantially purified nucleic acid molecule according to
claim 4, wherein said nucleic acid molecule further comprises a
bacterial ORI site.
6. The substantially purified nucleic acid molecule according to
claim 1, wherein said nucleic acid molecule has a promoter or
partial promoter region.
7. The substantially purified nucleic acid molecule according to
claim 6, wherein said promoter region comprises a CAAT cis element
and a TATA cis element and an additional cis element.
8. A substantially purified nucleic acid molecule comprising a
nucleic acid molecule or fragment thereof having a pair of defined
ends, wherein said pair of defined ends are selected from the
defined ends in Table A.
9. The substantially purified nucleic acid molecule according to
claim 8, wherein said molecule comprises a nucleic acid molecule
having one or two of said defined ends.
10. The substantially purified nucleic acid molecule according to
claim 9, wherein said molecule comprises a nucleic acid molecule
having two of said defined ends.
11. A substantially purified protein or fragment thereof encoded by
a first nucleic acid molecule which specifically hybridizes to a
second nucleic acid molecule, said second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of SEQ ID NO: 1 through SEQ ID NO: 36935 or complements
thereof.
12. A transformed plant having a nucleic acid molecule which
comprises: (A) an exogenous promoter region which functions in a
plant cell to cause the production of a mRNA molecule; which is
linked to (B) a structural nucleic acid molecule, wherein said
structural nucleic acid molecule is selected from the group
consisting of SEQ ID NO: 1 through SEQ ID NO: 36935 or complements
thereof or fragment of either; which is linked to (C) a 3'
non-translated sequence that functions in a plant cell to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of said mRNA molecule.
13. The transformed plant according to claim 12, wherein said
structural nucleic acid molecule is in the antisense
orientation.
14. The transformed plant according to claim 12, wherein said plant
is a dicot.
15. The transformed plant according to claim 12, wherein said plant
is a monocot.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation under 35 U.S.C. .sctn.
120 of U.S. application Ser. No. 09/421,106 filed Oct. 15, 1999,
herein incorporated by reference in its entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] This application contains a sequence listing, which is
contained on three identical CD-ROMs: two copies of the sequence
listing (Copy 1 and Copy 2) and a sequence listing Computer
Readable Form (CRF), all of which are herein incorporated by
reference. All three CD-ROMs each contain one file called "15598C
seq list.txt" which is 23,696,009 bytes in size (measured in
MS-DOS) and which was created on Dec. 20, 2005.
FIELD OF THE INVENTION
[0003] The present invention is in the field of plant genetics.
More specifically the invention relates to nucleic acid molecules
and nucleic acid molecules that contain markers, in particular,
single nucleotide polymorphism (SNP) and repetitive element
markers. In addition, the present invention provides nucleic acid
molecules having regulatory elements or encoding proteins or
fragments thereof. The invention also relates to proteins and
fragments of proteins so encoded and antibodies capable of binding
the proteins. The invention also relates to methods of using the
nucleic acid molecules, markers, repetitive elements and fragments
of repetitive elements, regulatory elements, proteins and fragments
of proteins.
BACKGROUND OF THE INVENTION
I. Sequence Tagged Connector Nucleic Acid Molecules and the
Bacterial Artificial Chromosomes (BACs) Containing these
Sequences.
[0004] Sequence tagged connectors, or STCs, are sequences of insert
data generated from both ends (at the vector-insert point) of a BAC
clone in a genomic library. These sequences, and BACs containing
these STC sequences, can be used, for example, for marker
development, genetic mapping or linkage analysis, marker assisted
breeding, and physical genome mapping (Venter, et al., Nature,
381:364-366 (1996), the entirety of which is herein incorporated by
reference; Choi and Wing, on the Worldwide web at
genome.clemson.edu/protocols2-nj.html July, 1998). STCs can
represent a copy of up to a full length of a mRNA transcript, a
promoter element or part of a promoter, can contain simple sequence
repeats (also called microsatellites) repetitive elements or
fragments of repetitive elements, other DNA markers, or any
combination thereof.
[0005] Markers have been used in genetic mapping which can be a
step in isolating a gene. Genetic mapping or linkage analysis is
based on the level at which markers and genes are co-inherited
(Rothwell, Understanding Genetics. 4.sup.th Ed., Oxford University
Press, New York, p. 703 (1988)). Statistical tests like chi-square
analysis can be used to test the randomness of segregation or
linkage (Kochert, The Rockefeller Foundation International Program
on Rice Biotechnology, University of Georgia, Athens, Ga., pp. 1-14
(1989), the entirety of which is herein incorporated by reference).
In linkage mapping, the proportion of recombinant individuals out
of the total mapping population provides the information for
determining the genetic distance between the loci (Young,
Encyclopedia of Agricultural Science, Vol. 3, pp. 275-282 (1994),
the entirety of which is herein incorporated by reference).
[0006] Classical mapping studies utilize easily observable, visible
traits instead of molecular markers. These visible traits are also
known as naked eye polymorphisms. These traits can be morphological
like plant height, fruit size, shape and color or physiological
like disease response, photoperiod sensitivity or crop maturity.
Visible traits are useful and are still in use because they
represent actual phenotypes and are easy to score without any
specialized lab equipment. By contrast, the other types of genetic
markers are arbitrary loci for use in linkage mapping and often not
associated to specific plant phenotypes (Young, Encyclopedia of
Agricultural Science, Vol. 3, pp. 275-282 (1994)). Many
morphological markers cause such large effects on phenotype that
they are undesirable in breeding programs. Many other visible
traits have the disadvantage of being developmentally regulated
(i.e., expressed only at certain stages; or in specific tissues and
organs). Often times, visible traits mask the effects of linked
minor genes making it nearly impossible to identify desirable
linkages for selection (Tanksely, et al., Biotech. 7:257-264
(1989), the entirety of which is herein incorporated by
reference).
[0007] Although a number of important agronomic characters are
controlled by loci having major effects on phenotype, many
economically important traits, such as yield and some forms of
disease resistance, are quantitative in nature. This type of
phenotypic variation in a trait is characterized by continuous,
normal distribution of phenotypic values in a particular population
(Beckmann and Soller, Oxford Surveys of Plant Molecular Biology,
Miffen. (ed.), Vol. 3, Oxford University Press, UK., pp. 196-250
(1986), the entirety of which is herein incorporated by reference).
Such traits are governed by a large number of loci, Quantitative
Trait Loci (QTL), each of which can make a small positive or
negative effect to the final phenotype value of the trait (Beckmann
and Soller, Oxford Surveys of Plant Molecular Biology, Miffen.
(ed.), Vol. 3, Oxford University Press, U.K., pp. 196-250 (1986)).
Loci contributing to such genetic variation are often termed minor
genes as opposed to major genes with large effects that follow a
Mendelian pattern of inheritance. Polygenic traits are also
predicted to follow a Mendelian type of inheritance, however the
contribution of each locus is expressed as an increase or decrease
in the final trait value.
[0008] Markers have been used in physical mapping studies with BAC
libraries made from plant genomes. Such mapping studies have been
carried out in rice (Kim et al., Genomics 34:213-218 (1996), the
entirety of which is herein incorporated by reference; Hang, Plant
Mol. Biol. 35:129-133 (1997), the entirety of which is herein
incorporated by reference; Zhang and Wing., Plant Mol. Bio.
35:115-127 (1997), the entirety of which is herein incorporated by
reference; Chen et al., Proc. Acad. Sci. (U.S.A.) 94:3431-3435
(1997), the entirety of which is herein incorporated by reference;
Wang et al., Plant J. 7:525-533 (1995), the entirety of which is
herein incorporated by reference), sorghum (Zwick et al., Genetics
148:1983-1992 (1998), the entirety of which is herein incorporated
by reference; Zhang, et al., Molecular Breeding 2:11-24 (1996), the
entirety of which is herein incorporated by reference), maize
(Chen, et al., Proc. Acad. Sci. (U.S.A.) 94:3431-3435 (1997), the
entirety of which is herein incorporated by reference), and
Arabidopsis (Kim, et al., Genomics 34:213-218 (1996), the entirety
of which is herein incorporated by reference).
[0009] Repetitive elements have been used in physical mapping in
cereals (Ananiev, et al., Proc. Acad. Sci. (U.S.A.) 95:13073-8
(1998), the entirety of which is herein incorporated by reference;
McLean et al., Mol Gen Genet. 253:687-694 (1997), the entirety of
which is herein incorporated by reference).
II. Sequence Comparisons
[0010] STCs and sequenced BACs can be compared, for example, to
sequences that encode promoters or proteins. These homologies can
be determined by similarity searches (Adams, et al., Science
252:1651-1656 (1991), the entirety of which is herein incorporated
by reference).
[0011] A characteristic feature of a DNA sequence is that it can be
compared with other DNA sequences. Sequence comparisons can be
undertaken by determining the similarity of the test or query
sequence with sequences in publicly available or propriety
databases ("similarity analysis") or by searching for certain
motifs ("intrinsic sequence analysis")(e.g., cis elements)(Coulson,
Trends in Biotechnology, 12:76-80 (1994), the entirety of which is
herein incorporated by reference; Birren, et al., Genome Analysis,
1:543-559 (1997), the entirety of which is herein incorporated by
reference).
[0012] Similarity analysis includes database search and alignment.
Examples of public databases include the DNA Database of Japan
(DDBJ)(on the Worldwide web at ddbj.nig.ac.jp/); Genebank (on the
Worldwide web at ncbi.nlm.nih.gov/web/Genbank/Index.htlm); and the
European Molecular Biology Laboratory Nucleic Acid Sequence
Database (EMBL) (on the Worldwide web at
ebi.ac.uk/ebi_docs/embl_db.html). A number of different search
algorithms have been developed, one example of which are the suite
of programs referred to as BLAST programs. There are five
implementations of BLAST, three designed for nucleotide sequences
queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein
sequence queries (BLASTP and TBLASTN) (Coulson, Trends in
Biotechnology, 12:76-80 (1994); Birren, et al., Genome Analysis,
1:543-559 (1997)).
[0013] BLASTN takes a nucleotide sequence (the query sequence) and
its reverse complement and searches them against a nucleotide
sequence database. BLASTN was designed for speed, not maximum
sensitivity, and may not find distantly related coding sequences.
BLASTX takes a nucleotide sequence, translates it in three forward
reading frames and three reverse complement reading frames, and
then compares the six translations against a protein sequence
database. BLASTX is useful for sensitive analysis of preliminary
(single-pass) sequence data and is tolerant of sequencing errors
(Gish and States, Nature Genetics, 3:266-272 (1993), the entirety
of which is herein incorporated by reference). BLASTN and BLASTX
may be used in concert for analyzing STC data (Coulson, Trends in
Biotechnology, 12:76-80 (1994); Birren, et al., Genome Analysis,
1:543-559 (1997)).
[0014] Given a coding nucleotide sequence and the protein it
encodes, it is often preferable to use the protein as the query
sequence to search a database because of the greatly increased
sensitivity to detect more subtle relationships. This is due to the
larger alphabet of proteins (20 amino acids) compared with the
alphabet of nucleic acid sequences (4 bases), where it is far
easier to obtain a match by chance. In addition, with nucleotide
alignments, only a match (positive score) or a mismatch (negative
score) is obtained, but with proteins, the presence of conservative
amino acid substitutions can be taken into account. Here, a
mismatch may yield a positive score if the non-identical residue
has physical/chemical properties similar to the one it replaced.
Various scoring matrices are used to supply the substitution scores
of all possible amino acid pairs. A general purpose scoring system
is the BLOSUM62 matrix (Henikoff and Henikoff, Proteins, 17:49-61
(1993), the entirety of which is herein incorporated by reference),
which is currently the default choice for BLAST programs. BLOSUM62
is tailored for alignments of moderately diverged sequences and
thus may not yield the best results under all conditions. Altschul,
J. Mol. Biol. 36:290-300 (1993), the entirety of which is herein
incorporated by reference, uses a combination of three matrices to
cover all contingencies. This may improve sensitivity, but at the
expense of slower searches. In practice, a single BLOSUM62 matrix
is often used but others (PAM40 and PAM250) may be attempted when
additional analysis is necessary. Low PAM matrices are directed at
detecting very strong but localized sequence similarities, whereas
high PAM matrices are directed at detecting long but weak
alignments between very distantly related sequences.
[0015] Homologues in other organisms are available that can be used
for comparative sequence analysis. Multiple alignments are
performed to study similarities and differences in a group of
related sequences. CLUSTAL W is a multiple sequence alignment
package available that performs progressive multiple sequence
alignments based on the method of Feng and Doolittle, J. Mol. Evol.
25:351-360 (1987), the entirety of which is herein incorporated by
reference. Each pair of sequences is aligned and the distance
between each pair is calculated; from this distance matrix, a guide
tree is calculated, and all of the sequences are progressively
aligned based on this tree. A feature of the program is its
sensitivity to the effect of gaps on the alignment; gap penalties
are varied to encourage the insertion of gaps in probable loop
regions instead of in the middle of structured regions. Users can
specify gap penalties, choose between a number of scoring matrices,
or supply their own scoring matrix for both the pairwise alignments
and the multiple alignments. CLUSTAL W for UNIX and VMS systems is
available at: ftp.ebi.ac.uk. Another program is MACAW (Schuler et
al., Proteins, Struct. Func. Genet, 9:180-190 (1991), the entirety
of which is herein incorporated by reference, for which both
Macintosh and Microsoft Windows versions are available. MACAW uses
a graphical interface, provides a choice of several alignment
algorithms, and is available by anonymous ftp at: ncbi.nlm.nih.gov
(directory/pub/macaw).
[0016] Sequence motifs are derived from multiple alignments and can
be used to examine individual sequences or an entire database for
subtle patterns. With motifs, it is sometimes possible to detect
distant relationships that may not be demonstrable based on
comparisons of primary sequences alone. Currently, the largest
collection of sequence motifs in the world is PROSITE (Bairoch and
Bucher, Nucleic Acid Research, 22:3583-3589 (1994), the entirety of
which is herein incorporated by reference). PROSITE may be accessed
via either the ExPASy server on the World Wide Web or anonymous ftp
site. Many commercial sequence analysis packages also provide
search programs that use PROSITE data.
[0017] A resource for searching protein motifs is the BLOCKS E-mail
server developed by S. Henikoff, Trends Biochem Sci., 18:267-268
(1993), the entirety of which is herein incorporated by reference;
Henikoff and Henikoff, Nucleic Acid Research, 19:6565-6572 (1991),
the entirety of which is herein incorporated by reference; Henikoff
and Henikoff, Proteins, 17:49-61 (1993). BLOCKS searches a protein
or nucleotide sequence against a database of protein motifs or
"blocks." Blocks are defined as short, ungapped multiple alignments
that represent highly conserved protein patterns. The blocks
themselves are derived from entries in PROSITE as well as other
sources. Either a protein or nucleotide query can be submitted to
the BLOCKS server; if a nucleotide sequence is submitted, the
sequence is translated in all six reading frames and motifs are
sought in these conceptual translations. Once the search is
completed, the server will return a ranked list of significant
matches, along with an alignment of the query sequence to the
matched BLOCKS entries.
[0018] Conserved protein domains can be represented by
two-dimensional matrices, which measure either the frequency or
probability of the occurrences of each amino acid residue and
deletions or insertions in each position of the domain. This type
of model, when used to search against protein databases, is
sensitive and usually yields more accurate results than simple
motif searches. Two popular implementations of this approach are
profile searches (such as GCG program ProfileSearch) and Hidden
Markov Models (HMMs) (Krough, et al., J. Mol. Biol. 235:1501-1531
(1994); Eddy, Current Opinion in Structural Biology 6:361-365
(1996), both of which are herein incorporated by reference in their
entirety). In both cases, a large number of common protein domains
have been converted into profiles, as present in the PROSITE
library, or HHM models, as in the Pfam protein domain library
(Sonnhammer, et al., Proteins 28:405-420 (1997), the entirety of
which is herein incorporated by reference). Pfam contains more than
500 HMM models for enzymes, transcription factors, signal
transduction molecules, and structural proteins. Protein databases
can be queried with these profiles or HMM models, which will
identify proteins containing the domain of interest. For example,
HMMSW or HMMFS, two programs in a public domain package called
HMMER (Sonnhammer, et al., Proteins 28:405-420 (1997)) can be
used.
[0019] PROSITE and BLOCKS represent collected families of protein
motifs. Thus, searching these databases entails submitting a single
sequence to determine whether or not that sequence is similar to
the members of an established family. Programs working in the
opposite direction compare a collection of sequences with
individual entries in the protein databases. An example of such a
program is the Motif Search Tool, or MoST (Tatusov, et al., Proc.
Natl. Acad. Sci. 91:12091-12095 (1994), the entirety of which is
herein incorporated by reference). On the basis of an aligned set
of input sequences, a weight matrix is calculated by using one of
four methods (selected by the user); a weight matrix is simply a
representation, position by position in an alignment, of how likely
a particular amino acid will appear. The calculated weight matrix
is then used to search the databases. To increase sensitivity,
newly found sequences are added to the original data set, the
weight matrix is recalculated, and the search is performed again.
This procedure continues until no new sequences are found.
SUMMARY OF THE INVENTION
[0020] The present invention includes and provides a substantially
purified nucleic acid molecule, the nucleic acid molecule capable
of specifically hybridizing to a second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of SEQ ID NO: 1 through SEQ ID NO: 36935 or complement or fragment
thereof.
[0021] The present invention provides a substantially purified
nucleic acid molecule comprising a nucleic acid molecule or
fragment thereof having a pair of defined ends, wherein the pair of
defined ends are selected from the defined ends in Table A.
[0022] The present invention provides a substantially purified
protein or fragment thereof encoded by a first nucleic acid
molecule which specifically hybridizes to a second nucleic acid
molecule, the second nucleic acid molecule having a nucleic acid
sequence selected from the group consisting of SEQ ID NO:1 through
SEQ ID NO:36935 or complements thereof.
[0023] The present invention provides a substantially purified
protein or fragment thereof encoded by a nucleic acid sequence
selected from the group consisting of SEQ ID NO:1 through SEQ ID
NO:36935 or complements thereof.
[0024] The present invention provides a transformed plant having a
nucleic acid molecule which comprises: (A) an exogenous promoter
region which functions in a plant cell to cause the production of a
mRNA molecule; which is linked to (B) a structural nucleic acid
molecule, wherein the structural nucleic acid molecule is selected
from the group consisting of SEQ ID NO:1 through SEQ ID NO:36935 or
complements thereof or fragments of either; which is linked to (C)
a 3' non-translated sequence that functions in a plant cell to
cause termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
[0025] The present invention provides a transformed plant having a
nucleic acid molecule which comprises: (A) an exogenous promoter
region which functions in a plant cell to cause the production of a
mRNA molecule wherein the promoter nucleic acid molecule is
selected from the group consisting of SEQ ID NO:1 through SEQ ID
NO:36935 or complements thereof or fragments of either; which is
linked to (B) a structural nucleic acid molecule encoding a protein
or peptide; which is linked to (C) a 3' non-translated sequence
that functions in a plant cell to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of the mRNA molecule.
[0026] The present invention provides a transformed plant having a
nucleic acid molecule which comprises: (A) an exogenous promoter
region which functions in a plant cell to cause the production of a
mRNA molecule; which is linked to (B) a transcribed nucleic acid
molecule with a transcribed strand and a non-transcribed strand,
wherein the transcribed strand is complementary to a nucleic acid
molecule having a nucleic acid sequence selected from the group
consisting of SEQ ID NO:1 through SEQ ID NO:36935 or complements
thereof or fragments of either and the transcribed strand is
complementary to an endogenous mRNA molecule; which is linked to
(C) a 3' non-translated sequence that functions in plant cells to
cause termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
[0027] The present invention provides a transformed plant having a
nucleic acid molecule which comprises: (A) an exogenous promoter
region which functions in a plant cell to cause the production of a
mRNA molecule wherein the promoter nucleic acid molecule is
selected from the group consisting of SEQ ID NO:1 through SEQ ID
NO:36935 or complements thereof or fragments of either; which is
linked to (B) a transcribed nucleic acid molecule with a
transcribed strand and a non-transcribed strand, wherein the
transcribed strand is complementary to an endogenous mRNA molecule;
which is linked to (C) a 3' non-translated sequence that functions
in plant cells to cause termination of transcription and addition
of polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
[0028] The present invention provides a computer readable medium
having recorded thereon one or more of the nucleotide sequences
depicted in SEQ ID NO:1 through SEQ ID NO: 36935.
[0029] The present invention provides a method of introgressing a
trait into a plant comprising using a nucleic acid marker for
marker assisted selection of the plant, the nucleic acid marker
complementary to a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1 through SEQ ID NO: 36935 or complements
thereof, and introgressing the trait into a plant.
[0030] The present invention provides a method for screening for a
trait comprising interrogating genomic DNA for the presence or
absence of a marker molecule that is genetically linked to a
nucleic acid sequence complementary to a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 36935 or complements thereof; and detecting the presence or
absence of the marker.
[0031] The present invention provides a method for determining the
likelihood of the level, presence or absence of a trait in a plant
comprising the steps of: (A) obtaining genomic DNA from the plant;
(B) detecting a marker nucleic acid molecule; the marker nucleic
acid molecule wherein the marker nucleic acid molecule specifically
hybridizes with a nucleic acid sequence that is genetically linked
to a nucleic acid sequence complementary to a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 36935 or complements thereof; (C) and determining the level,
presence or absence of the marker nucleic acid molecule, wherein
the level, presence or absence of the marker nucleic acid molecule
is indicative of the likely presence in the plant of the trait.
[0032] The present invention provides a method for determining a
genomic polymorphism in a plant that is predictive of a trait
comprising the steps: (A) incubating a marker nucleic acid
molecule, under conditions permitting nucleic acid hybridization,
and a complementary nucleic acid molecule obtained from the plant,
the marker nucleic acid molecule having a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 36935 or complements thereof; (B) permitting hybridization
between the marker nucleic acid molecule and the complementary
nucleic acid molecule obtained from the plant; and (C) detecting
the presence of the polymorphism.
[0033] The present invention provides a method of determining an
association between a polymorphism and a plant trait comprising:
(A) hybridizing a nucleic acid molecule specific for the
polymorphism to genetic material of a plant, wherein the nucleic
acid molecule comprises a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 1 through SEQ ID NO: 36935 or
complements thereof; and (B) calculating the degree of association
between the polymorphism and the plant trait.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Agents of the Invention:
[0035] (a) Nucleic Acid Molecules
[0036] Agents of the present invention include nucleic acid
molecules and more specifically BACs and STC nucleic acid molecules
or fragments thereof.
[0037] A subset of the nucleic acid molecules of the present
invention includes nucleic acid molecules that are marker
molecules. Another subset of the nucleic molecules of the present
invention include nucleic acid molecules that are promoters and/or
regulatory elements. Another subset of the nucleic acid molecules
of the present invention include nucleic acid molecules that encode
proteins or fragments of proteins. In a preferred embodiment the
nucleic acid molecules of the present invention are derived from
Glycine max (soybean) and more preferably Glycine max, genotype
A3244.
[0038] Fragment STC nucleic acid molecules and fragments of BACs
may encode significant portion(s) of, or indeed most of, the STC or
BAC nucleic acid molecule. In addition, a fragment nucleic acid
molecule can encode a Glycine max protein or fragment thereof.
Alternatively, the fragments may comprise smaller oligonucleotides
(having from about 15 to about 250 nucleotide residues, and more
preferably, about 15 to about 30 nucleotide residues).
[0039] The term "substantially purified", as used herein, refers to
a molecule separated from substantially all other molecules
normally associated with it in its native state. More preferably a
substantially purified molecule is the predominant species present
in a preparation. A substantially purified molecule may be greater
than 60% free, preferably 75% free, more preferably 90% free, and
most preferably 95% free from the other molecules (exclusive of
solvent) present in the natural mixture. The term "substantially
purified" is not intended to encompass molecules present in their
native state.
[0040] The agents of the present invention will preferably be
"biologically active" with respect to either a structural
attribute, such as the capacity of a nucleic acid to hybridize to
another nucleic acid molecule, or the ability of a protein to be
bound by an antibody (or to compete with another molecule for such
binding). Alternatively, such an attribute may be catalytic, and
thus involve the capacity of the agent to mediate a chemical
reaction or response.
[0041] The agents of the present invention may also be recombinant.
As used herein, the term recombinant means any agent (e.g., DNA,
peptide etc.), that is, or results, however indirect, from human
manipulation of a nucleic acid molecule.
[0042] It is understood that the agents of the present invention
may be labeled with reagents that facilitate detection of the agent
(e.g., fluorescent labels (Prober, et al., Science 238:336-340
(1987); Albarella et al., EP 144914, chemical labels (Sheldon et
al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No.
4,563,417, modified bases (Miyoshi et al., EP 119448, all of which
are hereby incorporated by reference in their entirety).
[0043] It is further understood, that the present invention
provides bacterial, viral, microbial, insect, fungal and plant
cells comprising the agents of the present invention. The BAC
nucleic acid molecules of the present invention include, without
limitation, BAC nucleic acid molecules having inserts with two
defined ends (STCs) as set forth in Table A. It is understood that
fragments of such BAC molecules can contain one or neither of the
defined ends.
[0044] STC nucleic acid molecules or fragment STC nucleic acid
molecules, or BACs or fragments thereof, of the present invention
are capable of specifically hybridizing to other nucleic acid
molecules under certain circumstances. As used herein, two nucleic
acid molecules are said to be capable of specifically hybridizing
to one another if the two molecules are capable of forming an
anti-parallel, double-stranded nucleic acid structure. A nucleic
acid molecule is said to be the "complement" of another nucleic
acid molecule if they exhibit complete complementarity. As used
herein, molecules are said to exhibit "complete complementarity"
when every nucleotide of one of the molecules is complementary to a
nucleotide of the other. Two molecules are said to be "minimally
complementary" if they can hybridize to one another with sufficient
stability to permit them to remain annealed to one another under at
least conventional "low-stringency" conditions. Similarly, the
molecules are said to be "complementary" if they can hybridize to
one another with sufficient stability to permit them to remain
annealed to one another under conventional "high-stringency"
conditions. Conventional stringency conditions are described by
Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd Ed.,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and by
Haymes, et al., Nucleic Acid Hybridization, A Practical Approach,
IRL Press, Washington, D.C. (1985), the entirety of which is herein
incorporated by reference. Departures from complete complementarity
are therefore permissible, as long as such departures do not
completely preclude the capacity of the molecules to form a
double-stranded structure. Thus, in order for an STC nucleic acid
molecule, fragment STC nucleic acid molecule, BAC nucleic acid
molecule or fragment BAC nucleic acid molecule to serve as a primer
or probe it need only be sufficiently complementary in sequence to
be able to form a stable double-stranded structure under the
particular solvent and salt concentrations employed.
[0045] Appropriate stringency conditions which promote DNA
hybridization are, for example, 6.0.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by a wash of
2.0.times.SSC at 50.degree. C., are known to those skilled in the
art or can be found in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt
concentration in the wash step can be selected from a low
stringency of about 2.0.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 50.degree. C. In addition, the
temperature in the wash step can be increased from low stringency
conditions at room temperature, about 22.degree. C., to high
stringency conditions at about 65.degree. C. Both temperature and
salt may be varied, or either the temperature or the salt
concentration may be held constant while the other variable is
changed.
[0046] In a preferred embodiment, a nucleic acid of the present
invention will specifically hybridize to one or more of the nucleic
acid molecules set forth in SEQ ID NO: 1 through SEQ ID NO: 36935
or complements thereof under moderately stringent conditions, for
example at about 2.0.times.SSC and about 40.degree. C.
[0047] In a particularly preferred embodiment, a nucleic acid of
the present invention will specifically hybridize to one or more of
the nucleic acid molecules set forth in SEQ ID NO: 1 through SEQ ID
NO: 36935 or complements thereof under high stringency conditions.
In one aspect of the present invention, the nucleic acid molecules
of the present invention have one or more of the nucleic acid
sequences set forth in SEQ ID NO: 1 through to SEQ ID NO: 36935 or
complements thereof. In another aspect of the present invention,
one or more of the nucleic acid molecules of the present invention
share between 100% and 90% sequence identity with one or more of
the nucleic acid sequences set forth in SEQ ID NO: 1 through to SEQ
ID NO: 36935 or complements thereof. In a further aspect of the
present invention, one or more of the nucleic acid molecules of the
present invention share between 100% and 95% sequence identity with
one or more of the nucleic acid sequences set forth in SEQ ID NO: 1
through to SEQ ID NO: 36935 or complements thereof. In a more
preferred aspect of the present invention, one or more of the
nucleic acid molecules of the present invention share between 100%
and 98% sequence identity with one or more of the nucleic acid
sequences set forth in SEQ ID NO: 1 through to SEQ ID NO: 36935 or
complements thereof. In an even more preferred aspect of the
present invention, one or more of the nucleic acid molecules of the
present invention share between 100% and 99% sequence identity with
one or more of the sequences set forth in SEQ ID NO: 1 through to
SEQ ID NO: 36935 or complements thereof. In a further, even more
preferred aspect of the present invention, one or more of the
nucleic acid molecules of the present invention exhibit 100%
sequence identity with one or more nucleic acid molecules present
within the genomic library herein designated BAC#1 (Monsanto
Company, St. Louis, Mo., United States of America).
[0048] It is understood that the present invention encompasses
fragments of such nucleic acid molecules and that such nucleic acid
fragments may contain one, part of one, or neither of the defined
sequences.
[0049] (a)(1) Nucleic Acid Molecule Markers
[0050] One aspect of the present invention concerns nucleic acid
molecules SEQ ID NO:1 through SEQ ID NO:36935 or complements
thereof, that contain microsatellites, single nucleotide
substitutions (SNPs), repetitive elements or parts of repetitive
elements or other markers. Microsatellites typically include a 1-6
nucleotide core element within SEQ ID NO:1 through SEQ ID NO:36935
that are tandemly repeated from one to many thousands of times. A
different "allele" occurs at an SSR locus as a result of changes in
the number of times a core element is repeated, altering the length
of the repeat region, (Brown et al., Methods of Genome Analysis in
Plants, (ed.) Jauhar, CRC Press, Inc, Boca Raton, Fla., USA;
London, England, UK, pp. 147-159, (1996), the entirety of which is
herein incorporated by reference). SSR loci occur throughout plant
genomes, and specific repeat motifs occur at different levels of
abundance than those found in animals. The relative frequencies of
all SSRs with repeat units of 1-6 nucleotides have been surveyed.
The most abundant SSR is AAAAAT followed by A.sub.n, AG.sub.n AAT,
AAC, AGC, AAG, AATT, AAAT and AC. On average, 1 SSR is found every
21 and 65 kb in dicots and monocots. Fewer CG nucleotides are found
in dicots than in monocots. There is no correlation between
abundance of SSRs and nuclear DNA content. The abundance of all tri
and tetranucleotide SSR combination jointly have been reported to
be equivalent to that of the total di-nucleotide combinations.
Mono- di- and tetra-nucleotide repeats are all located in noncoding
regions of DNA while 57% of those trinucleotide SSRs containing CG
were located within gene coding regions. All repeated trinucleotide
SSRs composed entirely of AT are found in noncoding regions, (Brown
et al., Methods of Genome Analysis in Plants, ed. Jauhar, CRC
Press, Inc, Boca Raton, Fla., USA; London, England, UK, pp.
147-159, (1996).
[0051] Microsatellites can be observed in SEQ NO:1 to SEQ NO: 36935
or complements thereof by using the BLASTN program to examine
sequences for the presence/absence of microsatellites. In this
system, raw sequence data is searched through databases, which
store SSR markers collected from publications and 692 classes of
di-, tri and tetranucleotide repeat markers generated by computer.
Microsatellites can also be observed by screening the BAC library
of the present invention by colony or plaque hybridization with a
labeled probe containing microsatellite markers; isolating positive
clones and sequencing the inserts of the positive clones; suitable
primers flanking the microsatellite markers.
[0052] Single nucleotide polymorphisms (SNPs) are single base
changes in genomic DNA sequence. They generally occur at greater
frequency than other markers and are spaced with a greater
uniformity throughout a genome than other reported forms of
polymorphism. The greater frequency and uniformity of SNPs means
that there is greater probability that such a polymorphism will be
found near or in a genetic locus of interest than would be the case
for other polymorphisms. SNPs are located in protein-coding regions
and noncoding regions of a genome. Some of these SNPs may result in
defective or variant protein expression (e.g., as a result of
mutations or defective splicing). Analysis (genotyping) of
characterized SNPs can require only a plus/minus assay rather than
a lengthy measurement, permitting easier automation.
[0053] SNPs can be characterized using any of a variety of methods.
Such methods include the direct or indirect sequencing of the site,
the use of restriction enzymes (Botstein et al., Am. J. Hum. Genet.
32:314-331 (1980), the entirety of which is herein incorporated
reference; Konieczny and Ausubel, Plant J. 4:403-410 (1993), the
entirety of which is herein incorporated by reference), enzymatic
and chemical mismatch assays (Myers et al., Nature 313:495-498
(1985), the entirety of which is herein incorporated by reference),
allele-specific PCR (Newton et al., Nucl. Acids Res. 17:2503-2516
(1989), the entirety of which is herein incorporated by reference;
Wu et al., Proc. Natl. Acad. Sci. USA 86:2757-2760 (1989), the
entirety of which is herein incorporated by reference), ligase
chain reaction (Barany, Proc. Natl. Acad. Sci. USA 88:189-193
(1991), the entirety of which is herein incorporated by reference),
single-strand conformation polymorphism analysis (Labrune et al.,
Am. J. Hum. Genet. 48: 1115-1120 (1991), the entirety of which is
herein incorporated by reference), primer-directed nucleotide
incorporation assays (Kuppuswami et al., Proc. Natl. Acad. Sci. USA
88:1143-1147 (1991), the entirety of which is herein incorporated
by reference), dideoxy fingerprinting (Sarkar et al., Genomics
13:441-443 (1992), the entirety of which is herein incorporated by
reference), solid-phase ELISA-based oligonucleotide ligation assays
(Nikiforov et al., Nucl. Acids Res. 22:4167-4175 (1994), the
entirety of which is herein incorporated by reference),
oligonucleotide fluorescence-quenching assays (Livak et al., PCR
Methods Appl. 4:357-362 (1995a), the entirety of which is herein
incorporated by reference), 5'-nuclease allele-specific
hybridization TaqMan.TM. assay (Livak et al., Nature Genet.
9:341-342 (1995), the entirety of which is herein incorporated by
reference), template-directed dye-terminator incorporation (TDI)
assay (Chen and Kwok, Nucl. Acids Res. 25:347-353 (1997), the
entirety of which is herein incorporated by reference),
allele-specific molecular beacon assay (Tyagi et al., Nature
Biotech. 16: 49-53 (1998), the entirety of which is herein
incorporated by reference), PinPoint assay (Haff and Smimov, Genome
Res. 7: 378-388 (1997), the entirety of which is herein
incorporated by reference), and dCAPS analysis (Neff et al., Plant
J. 14:387-392 (1998), the entirety of which is herein incorporated
by reference).
[0054] SNPs can be observed by examining sequences of overlapping
clones in the BAC library according to the method described by
Taillon-Miller et al. Genome Res. 8:748-754 (1998), the entirety of
which is herein incorporated). SNPs can also be observed by
screening the BAC library of the present invention by colony or
plaque hybridization with a labeled probe containing SNP markers;
isolating positive clones and sequencing the inserts of the
positive clones; suitable primers flanking the SNP markers.
[0055] Genetic markers of the present invention include "dominant"
or "codominant" markers. "Codominant markers" reveal the presence
of two or more alleles (two per diploid individual) at a locus.
"Dominant markers" reveal the presence of only a single allele per
locus. The presence of the dominant marker phenotype (e.g., a band
of DNA) is an indication that one allele is present in either the
homozygous or heterozygous condition. The absence of the dominant
marker phenotype (e.g., absence of a DNA band) is merely evidence
that "some other" undefined allele is present. In the case of
populations where individuals are predominantly homozygous and loci
are predominately dimorphic, dominant and codominant markers can be
equally valuable. As populations become more heterozygous and
multi-allelic, codominant markers often become more informative of
the genotype than dominant markers.
[0056] In addition to SSRs and SNPs, repetitive elements can be
used as markers. For most eukaryotes, interspersed repeat sequence
elements are typically mobile genetic elements (Wright et al.,
Genetics 142:569-578 (1996), the entirety of which is herein
incorporated by reference). They are ubiquitous in most living
organisms and are present in copy numbers ranging from just a few
elements to tens or hundreds or thousands per genome. In the latter
case, they can represent a major fraction of the genome. For
example, transposable elements have been estimated to make up
greater than 50% of the maize genome (Kidwell, and Lisch Proc.
Natl. Acad. Sci. (U.S.A.) 94:7704-7711 (1997), the entirety of
which is herein incorporated by reference).
[0057] Transposable elements are classified in families according
to their sequence similarity. Two major classes are distinguished
by their differing modes of transposition. Class I elements are
retroelements that use reverse transcriptase to transpose by means
of an RNA intermediate. They include long terminal repeat
retrotransposons and long and short interspersed elements (LINES
and SINES, respectively). Class II elements transpose directly from
DNA to DNA and include transposons such as the
Activator-Dissociation (Ac-Ds) family in maize, the P element in
Drosophila and the Tc-1 element in Caenhorabditis elegans.
Additionally, a category of transposable elements has been
discovered whose transposition mechanism is not yet known. These
miniature inverted-repeat transposable elements (MITEs) have some
properties of both class I and II elements. They are short (100-400
bp in length) and none so far has been found to have any coding
potential. They are present in high copy number (3,000-10,000) per
genome and have target site preferences for TAA or TA in plants
(Kidwell and Lisch, Proc. Natl. Acad. Sci. (U.S.A.) 94:7704-7711
(1997)).
[0058] Insertion elements are found in two areas of the genome.
Some are located in regions distant from gene sequences such as in
the heterochromatin or in regions between genes; other repeat
elements are found in or near single copy sequences. The insertion
of an Ac-Ds element into wx-m9, an allele of the waxy locus in
maize is an example of a repetitive element found within a coding
region. The effect of this insertion is attenuated by the loss
through splicing of the transposable element after transcription
(Kidwell and Lisch, Proc. Natl. Acad. Sci. (U.S.A.) 94:7704-7711
(1997)).
[0059] The genetic variability resulting from transposable elements
ranges from changes in the size and arrangement of whole genomes to
changes in single nucleotides. They may produce major effects on
phenotypic traits or small silent changes detectable only at the
DNA sequence level. Transposable elements may also produce
variation when they excise, leaving small footprints of their
previous presence (Kidwell and Lisch, Proc. Natl. Acad. Sci.
(U.S.A.) 94:7704-7711 (1997)).
[0060] In addition, other markers such as AFLP markers, RFLP
markers, RAPD markers, phenotypic markers or isozyme markers can be
utilized (Walton, Seed World 22-29 (July, 1993), the entirety of
which is herein incorporated by reference; Burow and Blake,
Molecular Dissection of Complex Traits, 13-29, Eds. Paterson, CRC
Press, New York (1988), the entirety of which is herein
incorporated by reference). DNA markers can be developed from
nucleic acid molecules using restriction endonucleases, the PCR
and/or DNA sequence information. RFLP markers result from single
base changes or insertions/deletions. These codominant markers are
highly abundant in plant genomes, have a medium level of
polymorphism and are developed by a combination of restriction
endonuclease digestion and Southern blotting hybridization. CAPS
are similarly developed from restriction nuclease digestion but
only of specific PCR products. These markers are also codominant,
have a medium level of polymorphism and are highly abundant in the
genome. The CAPS result from single base changes and
insertions/deletions. Another marker type, RAPDs, are developed
from DNA amplification with random primers and result from single
base changes and insertions/deletions in plant genomes. They are
dominant markers with a medium level of polymorphisms and are
highly abundant. AFLP markers require using the PCR on a subset of
restriction fragments from extended adapter primers. These markers
are both dominant and codominant, are highly abundant in genomes
and exhibit a medium level of polymorphism. SSRs require DNA
sequence information. These codominant markers result from repeat
length changes, are highly polymorphic, and do not exhibit as high
a degree of abundance in the genome as CAPS, AFLPs and RAPDs. SNPs
also require DNA sequence information. These codominant markers
result from single base substitutions. They are highly abundant and
exhibit a medium of polymorphism (Rafalski, et al., In:
Nonmammalian Genomic Analysis, ed. Birren and Lai, Academic Press,
San Diego, Calif., pp. 75-134 (1996), the entirety of which is
herein incorporated by reference). Methods to isolate such markers
are known in the art.
[0061] Long Terminal repeat retrotransposons and MITEs have been
found to be associated with the genes of many plants where some of
the transposable elements contribute regulatory sequences. MITEs
such as the Tourist element in maize and the Stowaway element in
Sorghum are found frequently in the 5' and 3' noncoding regions of
genes and are frequently associated with the regulatory regions of
genes of diverse flowering plants (Kidwell and Lisch, Proc. Natl.
Acad. Sci. (U.S.A.) 94:7704-7711 (1997)). It is understood that one
or more of the Long Terminal repeat retrotransposons and/or MITES
may be a marker, and even more preferably a marker for a gene.
[0062] (a)(2) Nucleic Acid Molecules Comprising Regulatory
Elements
[0063] Another class of agents of the present invention are nucleic
acid molecules having promoter regions or partial promoter regions
within SEQ ID NO: 1 through SEQ ID NO: 36935 or complements
thereof. Such promoter regions are typically found upstream of the
trinucleotide ATG sequence at the start site of a protein coding
region.
[0064] As used herein, a promoter region is a region of a nucleic
acid molecule that is capable, when located in cis to a nucleic
acid sequence that encodes for a protein or fragment thereof to
function in a way that directs expression of one or more mRNA
molecules that encodes for the protein or fragment thereof.
[0065] Promoters of the present invention can include between about
300 bp upstream and about 10 kb upstream of the trinucleotide ATG
sequence at the start site of a protein coding region. Promoters of
the present invention can preferably include between about 300 bp
upstream and about 5 kb upstream of the trinucleotide ATG sequence
at the start site of a protein coding region. Promoters of the
present invention can more preferably include between about 300 bp
upstream and about 2 kb upstream of the trinucleotide ATG sequence
at the start site of a protein coding region. Promoters of the
present invention can include between about 300 bp upstream and
about 1 kb upstream of the trinucleotide ATG sequence at the start
site of a protein coding region. While in many circumstances a 300
bp promoter may be sufficient for expression, additional sequences
may act to further regulate expression, for example, in response to
biochemical, developmental or environmental signals.
[0066] It is also preferred that the promoters of the present
invention contain a CAAT and a TATA cis element. Moreover, the
promoters of the present invention can contain one or more cis
elements in addition to a CAAT and a TATA box.
[0067] By "regulatory element" it is intended a series of
nucleotides that determines if, when, and at what level a
particular gene is expressed. The regulatory DNA sequences
specifically interact with regulatory or other proteins. Many
regulatory elements act in cis ("cis elements") and are believed to
affect DNA topology, producing local conformations that selectively
allow or restrict access of RNA polymerase to the DNA template or
that facilitate selective opening of the double helix at the site
of transcriptional initiation. Cis elements occur within, but are
not limited to promoters, and promoter modulating sequences
(inducible elements). Cis elements can be identified using known
cis elements as a target sequence or target motif in the BLAST
programs of the present invention.
[0068] Promoters of the present invention include homologues of cis
elements known to effect gene regulation that show homology with
the nucleic acid molecules of the present invention. These cis
elements include, but are not limited to, oxygen responsive cis
elements (Cowen, et al., J. Biol. Chem. 268(36):26904-26910 (1993)
the entirety of which is herein incorporated by reference), light
regulatory elements (Bruce and Quaill, Plant Cell 2 (11):1081-1089
(1990) the entirety of which is herein incorporated by reference;
Bruce, et al., EMBO J. 10:3015-3024 (1991), the entirety of which
is herein incorporated by reference; Rocholl, et al., Plant Sci.
97:189-198 (1994), the entirety of which is herein incorporated by
reference; Block, et al., Proc. Natl. Acad. Sci. (U.S.A.)
87:5387-5391 (1990), the entirety of which is herein incorporated
by reference; Giuliano, et al., Proc. Natl. Acad. Sci. (U.S.A.)
85:7089-7093 (1988), the entirety of which is herein incorporated
by reference; Staiger, et al., Proc. Natl. Acad. Sci. (U.S.A.)
86:6930-6934 (1989), the entirety of which is herein incorporated
by reference; Izawa, et al., Plant Cell 6:1277-1287 (1994), the
entirety of which is herein incorporated by reference; Menkens, et
al., Trends in Biochemistry 20:506-510 (1995), the entirety of
which is herein incorporated by reference; Foster, et al., FASEB J.
8:192-200 (1994), the entirety of which is herein incorporated by
reference; Plesse, et al., Mol Gen Gene 254:258-266 (1997), the
entirety of which is herein incorporated by reference; Green, et
al., EMBO J. 6:2543-2549 (1987), the entirety of which is herein
incorporated by reference; Kuhlemeier et al., Ann. Rev Plant
Physiol. 38:221-257 (1987), the entirety of which is herein
incorporated by reference; Villain et al., J. Biol. Chem.
271:32593-32598 (1996), the entirety of which is herein
incorporated by reference; Lam et al., Plant Cell 2:857-866 (1990),
the entirety of which is herein incorporated by reference;
Gilmartin, et al., Plant Cell 2:369-378 (1990), the entirety of
which is herein incorporated by reference; Datta, et al., Plant
Cell 1: 1069-1077 (1989) the entirety of which is herein
incorporated by reference; Gilmartin, et al., Plant Cell 2:369-378
(1990), the entirety of which is herein incorporated by reference;
Castresana, et al., EMBO J. 7:1929-1936 (1988), the entirety of
which is herein incorporated by reference; Ueda, et al., Plant Cell
1:217-227 (1989), the entirety of which is herein incorporated by
reference; Terzaghi, et al., Annu. Rev. Plant Physiol. Plant Mol.
Biol. 46:445-474 (1995), the entirety of which is herein
incorporated by reference; Green et al., EMBO J. 6:2543-2549
(1987), the entirety of which is herein incorporated by reference;
Villain, et al., J. Biol. Chem. 271:32593-32598 (1996), the
entirety of which is herein incorporated by reference; Tjaden, et
al., Plant Cell 6:107-118 (1994), the entirety of which is herein
incorporated by reference; Tjaden, et al., Plant Physiol.
108:1109-1117 (1995), the entirety of which is herein incorporated
by reference; Ngai, et al., Plant J. 12:1021-1234 (1997), the
entirety of which is herein incorporated by reference; Bruce, et
al., EMBO J. 10:3015-3024 (1991), the entirety of which is herein
incorporated by reference; Ngai, et al., Plant J. 12:1021-1034
(1997), the entirety of which is herein incorporated by reference),
elements responsive to gibberellin, (Muller, et al., J. Plant
Physiol. 145.606-613 (1995), the entirety of which is herein
incorporated by reference; Croissant, et al., Plant Science
116:27-35 (1996), the entirety of which is herein incorporated by
reference; Lohmer, et al., EMBO J. 10:617-624 (1991), the entirety
of which is herein incorporated by reference; Rogers, et al., Plant
Cell 4:1443-1451 (1992), the entirety of which is herein
incorporated by reference; Lanahan et al., Plant Cell 4:203-211
(1992) the entirety of which is herein incorporated by reference;
Skriver et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:7266-7270 (1991)
the entirety of which is herein incorporated by reference;
Gilmartin, et al., Plant Cell 2:369-378 (1990), the entirety of
which is herein incorporated by reference; Huang, et al., Plant
Mol. Biol. 14:655-668 (1990), the entirety of which is herein
incorporated by reference, Gubler, et al., Plant Cell 7:1879-1891
(1995), the entirety of which is herein incorporated by reference),
elements responsive to abscisic acid, (Busk, et al., Plant Cell
9:2261-2270 (1997), the entirety of which is herein incorporated by
reference; Guiltinan, et al., Science 250:267-270 (1990), the
entirety of which is herein incorporated by reference; Shen, et
al., Plant Cell 7:295-307 (1995) the entirety of which is herein
incorporated by reference; Shen et al., Plant Cell 8:1107-1119
(1996), the entirety of which is herein incorporated by reference;
Seo et al., Plant Mol. Biol. 27:1119-1131 (1995), the entirety of
which is herein incorporated by reference; Marcotte et al., Plant
Cell 1:969-976 (1989) the entirety of which is herein incorporated
by reference; Shen et al., Plant Cell 7:295-307 (1995), the
entirety of which is herein incorporated by reference; Iwasaki et
al., Mol Gen Genet 247:391-398 (1995), the entirety of which is
herein incorporated by reference; Hattori et al., Genes Dev.
6:609-618 (1992), the entirety of which is herein incorporated by
reference; Thomas et al., Plant Cell 5:1401-1410 (1993), the
entirety of which is herein incorporated by reference), elements
similar to abscisic acid responsive elements, (Ellerstrom et al.,
Plant Mol. Biol. 32:1019-1027 (1996), the entirety of which is
herein incorporated by reference), auxin responsive elements (Liu
et al., Plant Cell 6:645-657 (1994) the entirety of which is herein
incorporated by reference; Liu et al., Plant Physiol. 115:397-407
(1997), the entirety of which is herein incorporated by reference;
Kosugi et al., Plant J. 7:877-886 (1995), the entirety of which is
herein incorporated by reference; Kosugi et al., Plant Cell
9:1607-1619 (1997), the entirety of which is herein incorporated by
reference; Ballas et al., J. Mol. Biol. 233:580-596 (1993), the
entirety of which is herein incorporated by reference), a cis
element responsive to methyl jasmonate treatment (Beaudoin and
Rothstein, Plant Mol. Biol. 33:835-846 (1997), the entirety of
which is herein incorporated by reference), a cis element
responsive to abscisic acid and stress response (Straub et al.,
Plant Mol. Biol. 26:617-630 (1994), the entirety of which is herein
incorporated by reference), ethylene responsive cis elements
(Itzhaki et al., Proc. Natl. Acad. Sci. (U.S.A.) 91:8925-8929
(1994), the entirety of which is herein incorporated by reference;
Montgomery et al., Proc. Acad. Sci. (U.S.A.) 90:5939-5943 (1993),
the entirety of which is herein incorporated by reference; Sessa et
al., Plant Mol. Biol. 28:145-153 (1995), the entirety of which is
herein incorporated by reference; Shinshi et al., Plant Mol. Biol.
27:923-932 (1995), the entirety of which is herein incorporated by
reference), salicylic acid cis responsive elements, (Strange et
al., Plant J. 11:1315-1324 (1997), the entirety of which is herein
incorporated by reference; Qin et al., Plant Cell 6:863-874 (1994),
the entirety of which is herein incorporated by reference), a cis
element that responds to water stress and abscisic acid (Lam et
al., J. Biol. Chem. 266:17131-17135 (1991), the entirety of which
is herein incorporated by reference; Thomas et al., Plant Cell
5:1401-1410 (1993), the entirety of which is herein incorporated by
reference; Pla et al., Plant Mol Biol 21:259-266 (1993), the
entirety of which is herein incorporated by reference), a cis
element essential for M phase-specific expression (Ito et al.,
Plant Cell 10:331-341 (1998), the entirety of which is herein
incorporated by reference), sucrose responsive elements (Huang et
al., Plant Mol. Biol. 14:655-668 (1990), the entirety of which is
herein incorporated by reference; Hwang et al., Plant Mol Biol
36:331-341 (1998), the entirety of which is herein incorporated by
reference; Grierson et al., Plant J. 5:815-826 (1994), the entirety
of which is herein incorporated by reference), heat shock response
elements (Pelham et al., Trends Genet. 1:31-35 (1985), the entirety
of which is herein incorporated by reference), elements responsive
to auxin and/or salicylic acid and also reported for light
regulation (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.)
86:7890-7897 (1989), the entirety of which is herein incorporated
by reference; Benfey et al., Science 250:959-966 (1990), the
entirety of which is herein incorporated by reference), elements
responsive to ethylene and salicylic acid (Ohme-Takagi et al.,
Plant Mol. Biol. 15:941-946 (1990), the entirety of which is herein
incorporated by reference), elements responsive to wounding and
abiotic stress (Loake et al., Proc. Natl. Acad. Sci. (U.S.A.)
89:9230-9234 (1992), the entirety of which is herein incorporated
by reference; Mhiri et al., Plant Mol. Biol. 33:257-266 (1997), the
entirety of which is herein incorporated by reference), antioxidant
response elements (Rushmore et al., J. Biol. Chem. 266:11632-11639,
the entirety of which is herein incorporated by reference; Dalton
et al., Nucleic Acids Res. 22:5016-5023 (1994), the entirety of
which is herein incorporated by reference), Sph elements (Suzuki et
al., Plant Cell 9:799-807 1997), the entirety of which is herein
incorporated reference), Elicitor responsive elements, (Fukuda et
al., Plant Mol. Biol. 34:81-87 (1997), the entirety of which is
herein incorporated by reference; Rushton et al., EMBO J.
15:5690-5700 (1996), the entirety of which is herein incorporated
by reference), metal responsive elements (Stuart et al., Nature
317:828-831 (1985), the entirety of which is herein incorporated by
reference; Westin et al., EMBO J. 7:3763-3770 (1988), the entirety
of which is herein incorporated by reference; Thiele et al.,
Nucleic Acids Res. 20:1183-1191 (1992), the entirety of which is
herein incorporated by reference; Faisst et al., Nucleic Acids Res.
20:3-26 (1992), the entirety of which is herein incorporated by
reference), low temperature responsive elements, (Baker et al.,
Plant Mol. Biol. 24:701-713 (1994), the entirety of which is herein
incorporated by reference; Jiang et al., Plant Mol. Biol.
30:679-684 (1996), the entirety of which is herein incorporated by
reference; Nordin et al., Plant Mol. Biol. 21:641-653 (1993), the
entirety of which is herein incorporated by reference; Zhou et al.,
J. Biol. Chem. 267:23515-23519 (1992), the entirety of which is
herein incorporated by reference), drought responsive elements,
(Yamaguchi et al., Plant Cell 6:251-264 (1994), the entirety of
which is herein incorporated by reference; Wang et al., Plant Mol.
Biol. 28:605-617 (1995), the entirety of which is herein
incorporated by reference; Bray E A, Trends in Plant Science
2:48-54 (1997), the entirety of which is herein incorporated by
reference) enhancer elements for glutenin, (Colot et al., EMBO J.
6:3559-3564 (1987), the entirety of which is herein incorporated by
reference; Thomas et al., Plant Cell 2:1171-1180 (1990), the
entirety of which is incorporated by reference; Kreis et al.,
Philos. Trans. R. Soc. Lond., B314:355-365 (1986), the entirety of
which is herein incorporated by reference), light-independent
regulatory elements, (Lagrange et al., Plant Cell 9:1469-1479
(1997), the entirety of which is herein incorporated by reference;
Villain et al., J. Biol. Chem. 271:32593-32598 (1996), the entirety
of which is herein incorporated by reference), OCS enhancer
elements, (Bouchez et al., EMBO J. 8:4197-4204 (1989), the entirety
of which is herein incorporated by reference; Foley et al., Plant
J. 3:669-679 (1993), the entirety of which is herein incorporated
by reference), ACGT elements, (Foster et al., FASEB J. 8:192-200
(1994), the entirety of which is herein incorporated by reference;
Izawa et al., Plant Cell 6:1277-1287 (1994), the entirety of which
is herein incorporated by reference; Izawa et al., J. Mol. Biol.
230:1131-1144 (1993) the entirety of which is herein incorporated
by reference), negative cis elements in plastid related genes,
(Zhou et al., J. Biol. Chem. 267:23515-23519 (1992), the entirety
of which is herein incorporated by reference; Lagrange et al., Mol.
Cell. Biol. 13:2614-2622 (1993), the entirety of which is herein
incorporated by reference; Lagrange et al., Plant Cell 9:1469-1479
(1997), the entirety of which is herein incorporated by reference;
Zhou et al., J. Biol. Chem. 267:23515-23519 (1992), the entirety of
which is herein incorporated by reference), prolamin box elements,
(Forde et al., Nucleic Acids Res. 13:7327-7339 (1985), the entirety
of which is herein incorporated by reference; Colot et al., EMBO J.
6:3559-3564 (1987), the entirety of which is herein incorporated by
reference; Thomas et al., Plant Cell 2:1171-1180 (1990), the
entirety of which is herein incorporated by reference; Thompson et
al., Plant Mol. Biol. 15:755-764 (1990), the entirety of which is
herein incorporated by reference; Vicente et al., Proc. Natl. Acad.
Sci. (U.S.A.) 94:7685-7690 (1997), the entirety of which is herein
incorporated by reference), elements in enhancers from the IgM
heavy chain gene (Gillies et al., Cell 33:717-728 (1983), the
entirety of which is herein incorporated by reference; Whittier et
al., Nucleic Acids Res. 15:2515-2535 (1987), the entirety of which
is herein incorporated by reference.
[0069] (a)(3) Nucleic Acid Molecules Comprising Genes or Fragments
Thereof
[0070] Nucleic acid molecules of the present invention can comprise
one or more genes or fragments thereof. Such genes or fragments
thereof include homologues of known genes or protein coding regions
in other organisms or genes or fragments thereof that elicit only
limited or no matches with known genes or protein coding
regions.
[0071] Genomic sequences can be screened for the presence of
protein homologues or genes utilizing one or a number of different
search algorithms have that been developed, one example of which
are the suite of programs referred to as BLAST programs. Other
examples of suitable programs that can be utilized are known in the
art, several of which are described above in the Background and
under the section titled "Uses of the Agents of the Invention." In
addition, unidentified reading frames may be screened for protein
coding regions by prediction software such as GenScan, which is
located at the website gnomic.standford.edu/GENSCANW.html.
[0072] In a preferred embodiment of the present invention, the
Glycine max protein or fragment thereof of the present invention is
a homologue of another plant protein. In another preferred
embodiment of the present invention, the Glycine max protein or
fragment thereof of the present invention is a homologue of a
fungal protein. In another preferred embodiment of the present
invention, the Glycine max protein or fragment thereof of the
present invention is a homologue of a mammalian protein. In another
preferred embodiment of the present invention, the Glycine max
protein or fragment thereof of the present invention is a homologue
of a bacterial protein.
[0073] In a preferred embodiment of the present invention, the
Glycine max protein or fragments thereof or nucleic acid molecule
or fragment thereof has a BLAST score of more than 200, preferably
a BLAST score of more than 300, even more preferably a BLAST score
of more than 400.
[0074] In another preferred embodiment of the present invention,
the nucleic acid molecule encoding the Glycine max protein or
fragment thereof and/or nucleic acid molecule or fragment thereof
exhibits a % identity with its homologue of between about 25% and
about 40%, more preferably of between about 40 and about 70%, even
more preferably of between about 70% and about 90%, and even more
preferably between about 90% and 99%. In another preferred
embodiment, of the present invention, the Glycine max the nucleic
acid molecule encoding the Glycine max protein or fragment thereof
exhibits a % identity with its homologue of 100%.
[0075] In a preferred embodiment of the present invention, the
Glycine max protein or fragment thereof or nucleic acid molecule or
fragment thereof exhibits a % coverage of between about 0% and
about 33%, more preferably of between about 34% and about 66%, and
even more preferably of between about 67% and about 100%.
[0076] Genomic sequences can be screened for the presence of
proteins utilizing one or a number of different search algorithms
have that been developed, one example of which are the suite of
programs referred to as BLAST programs. Other examples of suitable
programs that can be utilized are known in the art, several of
which are described above in the Background. Nucleic acid molecules
of the present invention also include non-Glycine max homologues.
Preferred non-Glycine max homologues are selected from the group
consisting of alfalfa, Arabidopsis barley, Brassica, broccoli,
cabbage, citrus, cotton, garlic, oat, oilseed rape, onion, canola,
flax, an ornamental plant, maize, pea, peanut, pepper, potato,
rice, rye, sorghum, strawberry, sugarcane, sugarbeet, tomato,
wheat, poplar, pine, fir, eucalyptus, apple, lettuce, lentils,
grape, banana, tea, turf grasses, sunflower, oil palm, and
Phaseolus.
[0077] In a preferred embodiment, nucleic acid molecules having SEQ
ID NO: 1 through SEQ ID NO: 36935 or complements and fragments of
either can be utilized to obtain such homologues.
[0078] The degeneracy of the genetic code allows different nucleic
acid sequences to code for the same protein or peptide, e.g. see
U.S. Pat. No. 4,757,006, the entirety of which is herein
incorporated by reference. As used herein a nucleic acid molecule
is degenerate of another nucleic acid molecule when the nucleic
acid molecules encode for the same amino acid sequences but
comprise different nucleotide sequences. An aspect of the present
invention is that the nucleic acid molecules of the present
invention include nucleic acid molecules that are degenerate from
the STCs of this invention.
[0079] A further aspect of the present invention comprises one or
more nucleic acid molecules which differ in nucleic acid sequence
from those of a STC of this invention due to the degeneracy in the
genetic code in that they encode the same protein but differ in
nucleic acid sequence or a protein having one or more conservative
amino acid residue. Codons capable of coding for such conservative
substitutions are known in the art. For instance, serine is a
conservative substitute of alanine and threonine is a conservative
substitute for serine.
[0080] (a)(4) Nucleic Acid Molecules Comprising Introns and/or
Intron/Exon Junctions
[0081] Nucleic acid molecules of the present invention can comprise
an intron and/or one or more intron/exon junction. Sequences of the
present invention can be screened for introns and intron/exon
junctions utilizing one or a number of different search algorithms
that have that been developed, one example of which are the suite
of programs referred to as BLAST programs. Other examples of
suitable programs that can be utilized are known in the art,
several of which are described above in the Background and in the
section entitled "Uses of the Agents of the Present Invention".
[0082] (a)(5) Protein and Peptide Molecules
[0083] A class of agents comprises one or more of the protein or
peptide molecules encoded by SEQ ID NO: 1 through SEQ ID NO: 36935,
or complements thereof or fragments of either, or one or more of
the proteins encoded by a nucleic acid molecule or fragment thereof
or peptide molecules encoded by other nucleic acid agents of the
present invention. Protein and peptide molecules can be identified
using known protein or peptide molecules as a target sequence or
target motif in the BLAST programs of the present invention. In a
preferred embodiment, the protein or peptide molecules of the
present invention are derived from Glycine max (soybean) and more
preferably Glycine max, genotype A3244.
[0084] As used herein, the term "protein molecule" or "peptide
molecule" includes any molecule that comprises five or more amino
acids. It is well known in the art that proteins or peptides may
undergo modification, including post-translational modifications,
such as, but not limited to, disulfide bond formation,
glycosylation, phosphorylation, or oligomerization. Thus, as used
herein, the term "protein molecule" or "peptide molecule" includes
any protein molecule that is modified by any biological or
non-biological process. The terms "amino acid" and "amino acids"
refer to all naturally occurring L-amino acids. This definition is
meant to include norleucine, ornithine, homocysteine, and
homoserine.
[0085] One or more of the protein or fragments of peptide molecules
may be produced via chemical synthesis, or more preferably, by
expression in a suitable bacterial or eukaryotic host. Suitable
methods for expression are described by Sambrook, et al., Molecular
Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (1989), or similar texts.
[0086] A "protein fragment" is a peptide or polypeptide molecule
whose amino acid sequence comprises a subset of the amino acid
sequence of that protein. A protein or fragment thereof that
comprises one or more additional peptide regions not derived from
that protein is a "fusion" protein. Such molecules may be
derivatized to contain carbohydrate or other moieties (such as
keyhole limpet hemocyanin, etc.). Fusion protein or peptide
molecules of the present invention are preferably produced via
recombinant means.
[0087] Another class of agents comprises protein or peptide
molecules encoded by SEQ ID NO: 1 through SEQ ID NO: 36935 or
complements thereof or, fragments or fusions thereof in which
conservative, non-essential, or not relevant, amino acid residues
have been added, replaced, or deleted. An example of such a
homologue is the homologue protein of all non-Glycine max plant
species, including but not limited to alfalfa, barley, Brassica,
broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape,
onion, canola, flax, maize, an ornamental plant, pea, peanut,
pepper, potato, rice, rye, sorghum, strawberry, sugarcane,
sugarbeet, tomato, wheat, poplar, pine, fir, eucalyptus, apple,
lettuce, peas, lentils, grape, banana, tea, turf grasses, etc.
Particularly preferred non-Glycine max plants to utilize for the
isolation of homologues would include alfalfa, barley, cotton,
corn, oat, oilseed rape, rice, corn, canola, ornamentals,
sugarcane, sugarbeet, tomato, potato, wheat, and turf grasses. Such
a homologue can be obtained by any of a variety of methods. Most
preferably, as indicated above, one or more of the disclosed
sequences (SEQ ID NO: 1 through SEQ ID NO: 36935 or complements
thereof) will be used to define a pair of primers that may be used
to isolate the homologue-encoding nucleic acid molecules from any
desired species. Such molecules can be expressed to yield
homologues by recombinant means.
[0088] (a)(6) Antibodies
[0089] One aspect of the present invention concerns antibodies,
single-chain antigen binding molecules, or other proteins that
specifically bind to one or more of the protein or peptide
molecules of the present invention and their homologs, fusions or
fragments. Such antibodies may be used to quantitatively or
qualitatively detect the protein or peptide molecules of the
present invention. As used herein, an antibody or peptide is said
to "specifically bind" to a protein or peptide molecule of the
present invention if such binding is not competitively inhibited by
the presence of non-related molecules. In a preferred embodiment
the antibodies of the present invention bind to proteins of the
present invention, in a more preferred embodiment of the antibodies
of the present invention bind to proteins derived from Glycine
max.
[0090] Nucleic acid molecules that encode all or part of the
protein of the present invention can be expressed, via recombinant
means, to yield protein or peptides that can in turn be used to
elicit antibodies that are capable of binding the expressed protein
or peptide. Such antibodies may be used in immunoassays for that
protein. Such protein-encoding molecules, or their fragments may be
a "fusion" molecule (i.e., a part of a larger nucleic acid
molecule) such that, upon expression, a fusion protein is produced.
It is understood that any of the nucleic acid molecules of the
present invention may be expressed, via recombinant means, to yield
proteins or peptides encoded by these nucleic acid molecules.
[0091] The antibodies that specifically bind proteins and protein
fragments of the present invention may be polyclonal or monoclonal.
It is understood that practitioners are familiar with the standard
resource materials which describe specific conditions and
procedures for the construction, manipulation and isolation of
antibodies (see, for example, Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1988), the entirety of which is herein incorporated by
reference).
[0092] It is understood that any of the antibodies of the present
invention can be substantially purified and/or be biologically
active and/or recombinant.
[0093] Uses of the Agents of the Invention
[0094] Nucleic acid molecules of the present invention may be
employed to obtain other Glycine max nucleic acid molecules. Such
molecules can be readily obtained by using the above-described
nucleic acid molecules to screen libraries Glycine max
libraries.
[0095] Nucleic acid molecules and fragments thereof of the present
invention may also be employed to obtain nucleic acid molecule
homologs of non-Glycine max species including the nucleic acid
molecules that encode, in whole or in part, protein homologs of
other species or other organisms, sequences of genetic elements
such as promoters and transcriptional regulatory elements.
[0096] Nucleic acid molecules and fragments thereof of the present
invention may be employed for genetic mapping studies using linkage
analysis (genetic markers). A genetic linkage map shows the
relative locations of specific DNA markers along a chromosome. Maps
are used for the identification of genes associated with genetic
diseases or phenotypic traits, comparative genomics, and as a guide
for physical mapping. Through genetic mapping, a fine scale linkage
map can be developed using DNA markers, and, then, a genomic DNA
library of large-sized fragments can be screened with molecular
markers linked to the desired trait. In a preferred embodiment of
the present invention, the genomic library screened with the
nucleic acid molecules of the present invention is a genomic
library of Glycine max.
[0097] Mapping marker locations is based on the observation that
two markers located near each other on the same chromosome will
tend to be passed together from parent to offspring. During gamete
production, DNA strands occasionally break and rejoin in different
places on the same chromosome or on the homologous chromosome. The
closer the markers are to each other, the more tightly linked and
the less likely a recombination event will fall between and
separate them. Recombination frequency thus provides an estimate of
the distance between two markers.
[0098] In segregating populations, target genes have been reported
to have been placed within an interval of 5-10 cM with a high
degree of certainty (Tanksley et al., Trends in Genetics
11(2):63-68 (1995), the entirety of which is herein incorporated by
reference). The markers defining this interval are used to screen a
larger segregating population to identify individuals derived from
one or more gametes containing a crossover in the given interval.
Such individuals are useful in orienting other markers closer to
the target gene. Once identified, these individuals can be analyzed
in relation to all molecular markers within the region to identify
those closest to the target.
[0099] Markers of the present invention can be employed to
construct linkage maps and to locate genes with qualitative and
quantitative effects. The genetic linkage of additional marker
molecules can be established by a genetic mapping model such as,
without limitation, the flanking marker model reported by Lander
and Botstein, Genetics, 121:185-199 (1989), and the interval
mapping, based on maximum likelihood methods described by Lander
and Botstein, Genetics, 121:185-199 (1989), the entirety of which
is herein incorporated by reference and implemented in the software
package MAPMAKER/QTL (Lincoln and Lander, Mapping Genes Controlling
Quantitative Traits Using MAPMAKER/QTL, Whitehead Institute for
Biomedical Research, Massachusetts, (1990)). Additional software
includes Qgene, Version 2.23 (1996), Department of Plant Breeding
and Biometry, 266 Emerson Hall, Cornell University, Ithaca, N.Y.,
the manual of which is herein incorporated by reference in its
entirety). Use of the Qgene software is a particularly preferred
approach.
[0100] A maximum likelihood estimate (MLE) for the presence of a
marker is calculated, together with an MLE assuming no QTL effect,
to avoid false positives. A log.sub.10 of an odds ratio (LOD) is
then calculated as: LOD=log.sub.10 (MLE for the presence of a
QTL/MLE given no linked QTL).
[0101] The LOD score essentially indicates how much more likely the
data are to have arisen assuming the presence of a QTL than in its
absence. The LOD threshold value for avoiding a false positive with
a given confidence, say 95%, depends on the number of markers and
the length of the genome. Graphs indicating LOD thresholds are set
forth in Lander and Botstein, Genetics, 121:185-199 (1989), the
entirety of which is herein incorporated by reference and further
described by Arus and Moreno-Gonzalez, Plant Breeding, Hayward,
Bosemark, Romagosa (eds.) Chapman & Hall, London, pp. 314-331
(1993).
[0102] Additional models can be used. Many modifications and
alternative approaches to interval mapping have been reported,
including the use of non-parametric methods (Kruglyak and Lander,
Genetics, 139:1421-1428 (1995), the entirety of which is herein
incorporated by reference). Multiple regression methods or models
can be also be used, in which the trait is regressed on a large
number of markers (Jansen, Biometrics in Plant Breed, van Oijen,
Jansen (eds.) Proceedings of the Ninth Meeting of the Eucarpia
Section Biometrics in Plant Breeding, The Netherlands, pp. 116-124
(1994); Weber and Wricke, Advances in Plant Breeding, Blackwell,
Berlin, 16 (1994). Procedures combining interval mapping with
regression analysis, whereby the phenotype is regressed onto a
single putative QTL at a given marker interval, and at the same
time onto a number of markers that serve as `cofactors,` have been
reported by Jansen and Stam, Genetics, 136:1447-1455 (1994) and
Zeng, Genetics, 136:1457-1468 (1994). Generally, the use of
cofactors reduces the bias and sampling error of the estimated QTL
positions (Utz and Melchinger, Biometrics in Plant Breeding, van
Oijen, Jansen (eds.) Proceedings of the Ninth Meeting of the
Eucarpia Section Biometrics in Plant Breeding, The Netherlands, pp.
195-204 (1994), thereby improving the precision and efficiency of
QTL mapping (Zeng, Genetics, 136:1457-1468 (1994). These models can
be extended to multi-environment experiments to analysis
genotype-environment interactions (Jansen et al., Theo. Appl.
Genet. 91:33-37 (1995).
[0103] Selection of an appropriate mapping population is important
to map construction. The choice of appropriate mapping population
depends on the type of marker systems employed (Tanksley et al., J.
P. Gustafson and R. Appels (eds.), Plenum Press, New York, pp.
157-173 (1988), the entirety of which is herein incorporated by
reference). Consideration must be given to the source of parents
(adapted vs. exotic) used in the mapping population. Chromosome
pairing and recombination rates can be severely disturbed
(suppressed) in wide crosses (adapted.times.exotic) and generally
yield greatly reduced linkage distances. Wide crosses will usually
provide segregating populations with a relatively large array of
polymorphisms when compared to progeny in a narrow cross
(adapted.times.adapted).
[0104] An F.sub.2 population is the first generation of selfing
after the hybrid seed is produced. Usually a single F.sub.1 plant
is selfed to generate a population segregating for all the genes in
Mendelian (1:2:1) fashion. Maximum genetic information is obtained
from a completely classified F.sub.2 population using a codominant
marker system (Mather, Measurement of Linkage in Heredity: Methuen
and Co., (1938), the entirety of which is herein incorporated by
reference). In the case of dominant markers, progeny tests (e.g.,
F.sub.3, BCF.sub.2) are required to identify the heterozygotes,
thus making it equivalent to a completely classified F.sub.2
population. However, this procedure is often prohibitive because of
the cost and time involved in progeny testing. Progeny testing of
F.sub.2 individuals is often used in map construction where
phenotypes do not consistently reflect genotype (e.g., disease
resistance) or where trait expression is controlled by a QTL.
Segregation data from progeny test populations (e.g., F.sub.3 or
BCF.sub.2) can be used in map construction. Marker-assisted
selection can then be applied to cross progeny based on
marker-trait map associations (F.sub.2, F.sub.3), where linkage
groups have not been completely disassociated by recombination
events (i.e., maximum disequilibrium).
[0105] Recombinant inbred lines (RIL) (genetically related lines;
usually >F.sub.5, developed from continuously selfing F.sub.2
lines towards homozygosity) can be used as a mapping population.
Information obtained from dominant markers can be maximized by
using RIL because all loci are homozygous or nearly so. Under
conditions of tight linkage (i.e., about <10% recombination),
dominant and co-dominant markers evaluated in RIL populations
provide more information per individual than either marker type in
backcross populations (Reiter, Proc. Natl. Acad. Sci. (U.S.A.)
89:1477-1481 (1992). However, as the distance between markers
becomes larger (i.e., loci become more independent), the
information in RIL populations decreases dramatically when compared
to codominant markers.
[0106] Backcross populations (e.g., generated from a cross between
a successful variety (recurrent parent) and another variety (donor
parent) carrying a trait not present in the former) can be utilized
as a mapping population. A series of backcrosses to the recurrent
parent can be made to recover most of its desirable traits. Thus a
population is created consisting of individuals nearly like the
recurrent parent but each individual carries varying amounts or
mosaic of genomic regions from the donor parent. Backcross
populations can be useful for mapping dominant markers if all loci
in the recurrent parent are homozygous and the donor and recurrent
parent have contrasting polymorphic marker alleles (Reiter et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992). Information
obtained from backcross populations using either codominant or
dominant makers is less than that obtained from F.sub.2 populations
because one, rather than two, recombinant gametes are sampled per
plant. Backcross populations, however, are more informative (at low
marker saturation) when compared to RILs as the distance between
linked loci increases in RIL populations (i.e., about 0.15%
recombination). Increased recombination can be beneficial for
resolution of tight linkages, but may be undesirable in the
construction of maps with low marker saturation.
[0107] Near-isogenic lines (NIL)(created by many backcrosses to
produce an array of individuals that are nearly identical in
genetic composition except for the trait or genomic region under
interrogation) can be used as a mapping population. In mapping with
NILs, only a portion of the polymorphic loci are expected to map to
a selected region.
[0108] Bulk segregant analysis (BSA) is a method developed for the
rapid identification of linkage between markers and traits of
interest (Michelmore, et al., Proc. Natl. Acad. Sci. (U.S.A.)
88:9828-9832 (1991). In BSA, two bulked DNA samples are drawn from
a segregating population originating from a single cross. These
bulks contain individuals that are identical for a particular trait
(resistant or susceptible to particular disease) or genomic region
but arbitrary at unlinked regions (i.e., heterozygous). Regions
unlinked to the target region will not differ between the bulked
samples of many individuals in BSA.
[0109] Applications for markers in plant breeding include:
Quantitative Trait Loci (QTL) mapping (Edwards et al, Genetics
116:113-115 (1987), the entirety of which is herein incorporated by
reference); Nienhuis et al, Crop Sci. 27:797-803 (1987); Osborn et
al, Theor. Appl. Genet. 73:350-356 (1987); Romero-Severson et al,
Use of RFLPs In Analysis of Quantitative Trait Loci In Maize, In
Helentjaris and Burr (eds.) pp. 97-102 (1989), the entirety of
which is herein incorporated by reference; Young et al, Genetics
120:570-585 (1988), the entirety of which is herein incorporated by
reference; Martin et al, Science 243:1725-1728 (1989), the entirety
of which is herein incorporated by reference): Sarfatti et al.,
Theor. Appl Genet. 78:22-26 (1989), the entirety of which is herein
incorporated by reference; Tanksley, et al., Biotech. 7:257-264
(1989); Barone et al, Mol. Gen. Genet. 224:177-182 (1990), the
entirety of which is herein incorporated by reference); Jung et al,
Theor, Appl. Genet. 79:663-672 (1990), the entirety of which is
herein incorporated by reference; Keim et al, Genetics 126:735-742
(1990), the entirety of which is herein incorporated by reference,
Theor. Appl. Genet. 79:465-369 (1990), the entirety of which is
herein incorporated by reference; Paterson et al., Genetics
124:735-742 (1990), the entirety of which is herein incorporated by
reference; Martin et al, Proc. Natl. Acad. Sci. (U.S.A.)
88:2336-2340 (1991), the entirety of which is herein incorporated
by reference; Messeguer et al, Theor. Appl. Genet. 82:529-536
(1991), the entirety of which is herein incorporated by reference;
Michelmore et al, Proc Natl. Acad. Sci. (U.S.A.) 88:9828-9832
(1991), the entirety of which is herein incorporated by reference;
Ottaviano et al, Theor. Appl. Genet. 81:713-719 (1991), the
entirety of which is herein incorporated by reference; Yu et al,
Theor. Appl. Genet. 81:471-476 (1991), the entirety of which is
herein incorporated by reference; Diers et al, Crop Sci. 32:77-383
(1992), the entirety of which is herein incorporated by reference,
Theor. Appl. Genet. 83:608-612 (1992), the entirety of which is
herein incorporated by reference, J. Plant Nut. 15:2127-2136
(1992), the entirety of which is herein incorporated by reference;
Doebley et al, Proc. Natl. Acad. Sci. (U.S.A.) 87:9888-9892 (1990),
the entirety of which is herein incorporated by reference),
screening genetic resource strains for useful quantitative trait
alleles and introgression of these alleles into commercial
varieties (Beckmann and Soller, Theor. Appl. Genet. 67:35-43
(1983), the entirety of which is herein incorporated by reference;
Tanksley et al, (1989) the entirety of which is incorporated by
reference), or the mapping of mutations (Rafalski, et al., In.
Nonmammalian Genomic Analysis, ed. Birren and Lai, Academic Press,
San Diego, Calif., pp. 75-134 (1996). Additionally, markers can be
used to characterize transformants or germplasm, as a genetic
diagnostic test for plant breeding or to identify individuals or
varieties (Soller and Beckmann, Theor. Appl. Genet. 67:25-33
(1983), the entirety of which is herein incorporated by reference;
Tanksley et al, 1989). Markers also can be used to obtain
information about: (1) the number, effect, and chromosomal location
of each gene affecting a trait; (2) effects of multiple copies of
individual genes (gene dosage); (3) interaction between/among genes
controlling a trait (epistasis); (4) whether individual genes
affect more than one trait (pleiotropy); and (5) stability of gene
function across environments (G.times.E interactions).
[0110] It is understood that one or more of the nucleic acid
molecules of the present invention may in one embodiment be used as
markers in genetic mapping. In a preferred embodiment, nucleic acid
molecules of the present invention may in one embodiment be used as
markers with Glycine max.
[0111] The nucleic acid molecules of the present invention may be
used for physical mapping. Physical mapping, in conjunction with
linkage analysis, can enable the isolation of genes. Physical
mapping has been reported to identify the markers closest in terms
of genetic recombination to a gene target for cloning. Once a DNA
marker is linked to a gene of interest, the chromosome walking
technique can be used to find the genes via overlapping clones. For
chromosome walking, random molecular markers or established
molecular linkage maps are used to conduct a search to localize the
gene adjacent to one or more markers. A chromosome walk (Bukanov
and Berg, Mo. Microbiol, 11:509-523 (1994), the entirety of which
is herein incorporated by reference; Birkenbihl and Vielmetter
Nucleic Acids Res. 17:5057-5069 (1989), the entirety of which is
herein incorporated by reference; Wenzel and Herrmann, Nucleic
Acids Res. 16:8323-8336, (1988), the entirety of which is herein
incorporated by reference) is then initiated from the closest
linked marker. Starting from the selected clones, labeled probes
specific for the ends of the insert DNA are synthesized and used as
probes in hybridizations against a representative library. Clones
hybridizing with one of the probes are picked and serve as
templates for the synthesis of new probes; by subsequent analysis,
contigs are produced.
[0112] The degree of overlap of the hybridizing clones used to
produce a contig can be determined by comparative restriction
analysis. Comparative restriction analysis can be carried out in
different ways all of which exploit the same principle; two clones
of a library are very likely to overlap if they contain a limited
number of restriction sites for one or more restriction
endonucleases located at the same distance from each other. The
most frequently used procedures are, fingerprinting (Coulson et al,
Proc. Natl. Acad. Sci. (U.S.A.) 83:7821-7821, (1986), the entirety
of which is herein incorporated by reference); Knott et al.,
Nucleic Acids Res. 16:2601-2612 (1988), the entirety of which is
herein incorporated by reference; Eiglmeier et al., Mol. Microbiol.
7(2): 197-206 (1993), the entirety of which is herein incorporated
by reference, 1993), restriction fragment mapping (Smith and
Birnstiel, Nucleic Acids Res. 3:2387-2398 (1976), the entirety of
which is herein incorporated by reference, or the "landmarking"
technique (Charlebois et al., J. Mol. Biol. 222:509-524 (1991), the
entirety of which is herein incorporated by reference
[0113] To generate a physical map of a genome with BACs using the
fingerprinting technique, a BAC library containing a number of
clones equivalent to 4.times.-20.times. haploid genome can be used
(Zhang and Wing., Plant Mol. Bio. 35:115-127 (1997)). For example,
BAC DNA can be purified with the conventional alkaline lysis
procedure as used for plasmid DNA purification, digested with the
restriction enzyme used for construction of the BAC libraries and
end-labeled with .sup.32P-dATP, digested with Sau3AI and
fractionated on a denaturing polyacrylamide gel. The gel is dried
to chromatography paper and exposed to X-ray film. Fingerprints are
scanned and then converted into database records, according to the
positions of each band relative to the bands of the closest
molecular-weight marker on a gel. The incoming database of
fingerprints are first compared against each other to assemble
contigs if overlapped, and then compared against all existing
databases to place the incoming BACs and BAC contigs in established
contigs if overlapped. The physical length of a contig in kb is
estimated according to the number of restriction sites of the
enzyme used for the first digestion prior to fragment end labeling
Restriction analysis of a certain clone can be carried out, for
example, according to a method originally described by Smith and
Berstiel, Nucleic Acids Res. 3:2387-2398 (1976), First, the number
and size of cloned restriction fragments to be mapped are
determined by complete digestion and agarose gel electrophoresis.
Then, the clone is linearized at a unique restriction site outside
of the cloned DNA. Aliquots of the linearized molecules are
digested to different extents with the enzyme selected for mapping.
These partially cut samples are separated on agarose gels, blotted,
and hybridized to a labeled fragment of vector DNA. This probe is
derived entirely from one side or the other of the unique site used
to linearize the clone.
[0114] The results show a ladder of DNA fragments that have the
same unique end. By repeating these analyses in pairs with all the
neighboring intermediate DNA fragments, the correct order of
restriction fragments as well as the orientation of the cloned
insert can be deduced. The order of restriction fragments produced
by restriction enzymes other than the cloning enzyme can be
determined similarly. Fragment data from different enzymes are then
combined by a computer program and compared with the alignments of
other clones of the library (Kohara et al., Cell 50:495-508 (1987),
the entirety of which is herein incorporated by reference).
[0115] The landmarking technique can be carried out without any
labeling and relies on agarose gel analysis. Clones are first
digested preferably with a 6 bp specific endonuclease A, if
possible with the original clone enzyme. Clones are then digested
with a second endonuclease B. Endonuclease B is chosen based on its
ability to cut rarely in the genome, for example, on average only
once in 30 kbp. Of the fragments generated by digestion of one
clone with enzyme A, statistically only a small number (between
zero and three fragments) will also be cut by enzyme B. The very
specific pattern of those fragments which are produced by double
digestion are easily recognized. Any of these fragments which have
a restriction site for the rarely cutting endonuclease is called a
"landmark" Generally one common landmark is sufficient for defining
two overlapping clones.
[0116] Alternatively to chromosome walking and the associated
comparative restriction analyses methods, chromosome landing also
has been reported to be used to locate a gene of interest (Tanksley
et al., Trends in Genetics 11(2):63-68 (1995), the entirety of
which is herein incorporated by reference. For chromosome landing,
a DNA marker is isolated at a physical distance from the targeted
gene. High resolution linkage analysis is used to identify such a
marker that cosegregates with the gene. The marker is isolated at a
distance that is less than the average insert size of the genomic
library used for clone isolation. The DNA marker is then used to
screen the library and isolate (or "land" on) the clone containing
the gene without chromosome walking. Genome coverage of a library
can also be determined by cross-hybridization of individual large
insert clones by screening a BAC library with single copy RFLP
markers distributed randomly across the genome by hybridization. To
assure accuracy of the physical map, the markers should be
single-copy or of single-locus origin, if multiple-copy.
[0117] Chromosome landing of large-insert clones using
chromosome-specific DNA markers such as STSs microsatellites,
RFLPs, or other markers can correlate physical and genetic maps
(Zwick et al., Genetics 148:1983-1992 (1998), the entirety of which
is herein incorporated by reference in its entirety). These
strategies include chromosome landing of BACs containing markers or
BAC contigs by BAC-FISH (Fluorescent In Situ Hybridization), a
technique that involves tagging the DNA marker with an observable
label. BAC clones giving positive hybridization signals are
individually analyzed by FISH to metaphase chromosome spreads. The
location of the labeled probe can be detected after it binds to its
complementary DNA strand in an intact chromosome. The FISH of a BAC
selected from a BAC contig will directly place the BAC contig to a
specific chromosome region and establish a linkage relationships of
the BAC contig to another BAC contig.
[0118] Likewise, BACs and STCs of the present invention can be used
for contig mapping (Venter, et al., Nature, 381:364-366 (1996), the
entirety of which is herein incorporated by reference). A "seed"
BAC insert can be sequenced and then STCs and the corresponding BAC
of each STC can be placed on the sequenced insert using the BLASTN
program. Marker or gene containing STCs can be determined by the
BLASTN program and their corresponding BACs can be hybridized to
specific chromosomes using BAC-FISH (Zwick et al., Genetics
148:1983-1992 (1998)).
[0119] STCs can be used to identify a minimum tiling path of BACs
by computational procedures. Any nucleation sequence (the sequence
of an entire BAC, for example) can be electronically compared to a
database of STCs to identify the next clones to be sequenced to
maximally extend a contig. Chosen STCs need to occupy correct
positions in the tiling path. Several factors can contribute to
errors in the positioning and selection of these clones. An STC
that contains all or part of a repetitive element can appear to
align at any part of the growing mosaic which contains that
element. One method of selecting the appropriate BAC is to mask out
all sections of DNA sequence which are known to be repetitive
elements. The sequence symbols of these section are replaced with
Ns. These sections of DNA are not used to align the STC. STCs which
are completely comprised of Ns are discarded. In this way, the
unmasked sections of DNA may be aligned against the growing mosaic
without misplacing them due to redundant sequence. A program
publicly available, PowerBLAST includes a number of options for
masking repetitive elements and low complexity subsequences (Zhang
and Madden, Genome Res 7.649-56 (1997), the entirety of which is
herein incorporated by reference. cDNA and genomic libraries also
can be used as probe sources, thus directly combining the ordering
of the genomic DNA with the localization of transcribed sequences.
By a simultaneous hybridization to the genomic and back to the
transcriptional libraries, results are produced on sequence
homologies between transcribed sequences.
[0120] It is understood that the nucleic acid molecules of the
present invention may in one embodiment be used in physical
mapping. In a preferred embodiment, nucleic acid molecules of the
present invention may in one embodiment be used in the physical
mapping of Glycine max.
[0121] Nucleic acid molecules of the present invention can be used
in comparative mapping (physical and genetic). Comparative mapping
within families provides a method to the degree of sequence
conservation, gene order, ploidy of species, ancestral
relationships and the rates at which individual genomes are
evolving. Comparative mapping has been carried out by
cross-hybridizing molecular markers across species within a given
family. As in genetic mapping, molecular markers are needed but
instead of direct hybridization to mapping filters, the markers are
used to select large insert clones from a total genomic DNA library
of a related species. The selected clones, each a representative of
a single marker, can then be used to physically map the region in
the target species. The advantage of this method for comparative
mapping is that no mapping population or linkage map of the target
species is needed and the clones may also be used in other closely
related species. By comparing the results obtained by genetic
mapping in model plants, with those from other species,
similarities of genomic structure among plants species can be
established. Cross-hybridization of RFLP markers have been reported
and conserved gene order has been established in many studies. Such
macroscopic synteny is utilized for the estimation of
correspondence of loci among these crops. These loci include not
only Mendelian genes but also Quantitative Trait Loci (QTL) (Mohan
et al., Molecular Breeding 3:87-103 (1997), the entirety of which
is herein incorporated by reference.
[0122] It is understood that markers of the present invention may
in another embodiment be used in comparative mapping. In a
preferred embodiment the markers of present invention may be used
in the comparative mapping of Glycine clandestina, Glycine
gracilis, Glycine soja, Glycine tomentella, and Glycine
tabaina.
[0123] The nucleic acid molecules of the present invention can be
used to identify polymorphisms. In one embodiment, one or more of
the STC nucleic acid molecules or a BAC nucleic acid molecule (or a
sub-fragment of either) may be employed as a marker nucleic acid
molecule to identify such polymorphism(s). Alternatively, such
polymorphisms can be detected through the use of a marker nucleic
acid molecule or a marker protein that is genetically linked to
(i.e., a polynucleotide that co-segregates with) such
polymorphism(s).
[0124] In an alternative embodiment, such polymorphisms can be
detected through the use of a marker nucleic acid molecule that is
physically linked to such polymorphism(s). For this purpose, marker
nucleic acid molecules comprising a nucleotide sequence of a
polynucleotide located within 1 mb of the polymorphism(s), and more
preferably within 100 kb of the polymorphism(s), and most
preferably within 10 kb of the polymorphism(s) can be employed.
[0125] The genomes of animals and plants naturally undergo
spontaneous mutation in the course of their continuing evolution
(Gusella, Ann. Rev. Biochem. 55:831-854 (1986)). A "polymorphism"
is a variation or difference in the sequence of the gene or its
flanking regions that arises in some of the members of a species.
The variant sequence and the "original" sequence co-exist in the
species' population. In some instances, such co-existence is in
stable or quasi-stable equilibrium.
[0126] A polymorphism is thus said to be "allelic," in that, due to
the existence of the polymorphism, some members of a species may
have the original sequence (i.e., the original "allele") whereas
other members may have the variant sequence (i.e., the variant
"allele"). In the simplest case, only one variant sequence may
exist, and the polymorphism is thus said to be di-allelic. In other
cases, the species' population may contain multiple alleles, and
the polymorphism is termed tri-allelic, etc. A single gene may have
multiple different unrelated polymorphisms. For example, it may
have a di-allelic polymorphism at one site, and a multi-allelic
polymorphism at another site.
[0127] The variation that defines the polymorphism may range from a
single nucleotide variation to the insertion or deletion of
extended regions within a gene. In some cases, the DNA sequence
variations are in regions of the genome that are characterized by
short tandem repeats (STRs) that include tandem di- or
tri-nucleotide repeated motifs of nucleotides. Polymorphisms
characterized by such tandem repeats are referred to as "variable
number tandem repeat" ("VNTR") polymorphisms. VNTRs have been used
in identity analysis (Weber, U.S. Pat. No. 5,075,217; Armour, et
al., FEBS Lett. 307:113-115 (1992); Jones, et al., Eur. J.
Haematol. 39:144-147 (1987); Horn, et al., PCT Application
WO91/14003; Jeffreys, European Patent Application 370,719;
Jeffreys, U.S. Pat. No. 5,175,082; Jeffreys et al., Amer. J. Hum.
Genet. 39:11-24 (1986); Jeffreys et al., Nature 316:76-79 (1985);
Gray, et al., Proc. R. Acad. Soc. Lond. 243:241-253 (1991); Moore,
et al., Genomics 10:654-660 (1991); Jeffreys, et al., Anim. Genet.
18:1-15 (1987); Hillel, et al., Anim. Genet. 20:145-155 (1989);
Hillel, et al., Genet. 124:783-789 (1990), all of which are herein
incorporated by reference in their entirety).
[0128] The detection of polymorphic sites in a sample of DNA may be
facilitated through the use of nucleic acid amplification methods.
Such methods specifically increase the concentration of
polynucleotides that span the polymorphic site, or include that
site and sequences located either distal or proximal to it. Such
amplified molecules can be readily detected by gel electrophoresis
or other means.
[0129] The most preferred method of achieving such amplification
employs the polymerase chain reaction ("PCR") (Mullis, et al., Cold
Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich, et al.,
European Patent Appln. 50,424; European Patent Appln. 84,796,
European Patent Application 258,017, European Patent Appln.
237,362; Mullis, European Patent Appln. 201,184; Mullis, et al.,
U.S. Pat. No. 4,683,202; Erlich., U.S. Pat. No. 4,582,788; and
Saiki, et al., U.S. Pat. No. 4,683,194, all of which are herein
incorporated by reference), using primer pairs that are capable of
hybridizing to the proximal sequences that define a polymorphism in
its double-stranded form.
[0130] In lieu of PCR, alternative methods, such as the "Ligase
Chain Reaction" ("LCR") may be used (Barany, Proc. Natl. Acad. Sci.
(U.S.A.) 88:189-193 (1991), the entirety of which is herein
incorporated by reference. LCR uses two pairs of oligonucleotide
probes to exponentially amplify a specific target. The sequences of
each pair of oligonucleotides is selected to permit the pair to
hybridize to abutting sequences of the same strand of the target.
Such hybridization forms a substrate for a template-dependent
ligase. As with PCR, the resulting products thus serve as a
template in subsequent cycles and an exponential amplification of
the desired sequence is obtained.
[0131] LCR can be performed with oligonucleotides having the
proximal and distal sequences of the same strand of a polymorphic
site. In one embodiment, either oligonucleotide will be designed to
include the actual polymorphic site of the polymorphism. In such an
embodiment, the reaction conditions are selected such that the
oligonucleotides can be ligated together only if the target
molecule either contains or lacks the specific nucleotide that is
complementary to the polymorphic site present on the
oligonucleotide. Alternatively, the oligonucleotides may be
selected such that they do not include the polymorphic site (see,
Segev, PCT Application WO 90/01069, the entirety of which is herein
incorporated by reference).
[0132] The "Oligonucleotide Ligation Assay" ("OLA") may
alternatively be employed (Landegren, et al., Science 241:1077-1080
(1988), the entirety of which is herein incorporated by reference).
The OLA protocol uses two oligonucleotides which are designed to be
capable of hybridizing to abutting sequences of a single strand of
a target. OLA, like LCR, is particularly suited for the detection
of point mutations. Unlike LCR, however, OLA results in "linear"
rather than exponential amplification of the target sequence.
[0133] Nickerson, et al. have described a nucleic acid detection
assay that combines attributes of PCR and OLA (Nickerson, et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990), the entirety
of which is herein incorporated by reference). In this method, PCR
is used to achieve the exponential amplification of target DNA,
which is then detected using OLA. In addition to requiring
multiple, and separate, processing steps, one problem associated
with such combinations is that they inherit all of the problems
associated with PCR and OLA.
[0134] Schemes based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide, are also known (Wu, et al., Genomics 4:560
(1989), the entirety of which is herein incorporated by reference),
and may be readily adapted to the purposes of the present
invention.
[0135] Other known nucleic acid amplification procedures, such as
allele-specific oligomers, branched DNA technology,
transcription-based amplification systems, or isothermal
amplification methods may also be used to amplify and analyze such
polymorphisms (Malek, et al., U.S. Pat. No. 5,130,238; Davey, et
al., European Patent Application 329,822; Schuster et al., U.S.
Pat. No. 5,169,766; Miller, et al., PCT Application WO 89/06700;
Kwoh, et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1173-1177 (1989);
Gingeras, et al., PCT Application WO 88/10315; Walker, et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992), all of which are
herein incorporated by reference in their entirety).
[0136] The identification of a polymorphism can be determined in a
variety of ways. By correlating the presence or absence of it in an
plant with the presence or absence of a phenotype, it is possible
to predict the phenotype of that plant. If a polymorphism creates
or destroys a restriction endonuclease cleavage site, or if it
results in the loss or insertion of DNA (e.g., a VNTR
polymorphism), it will alter the size or profile of the DNA
fragments that are generated by digestion with that restriction
endonuclease. As such, individuals that possess a variant sequence
can be distinguished from those having the original sequence by
restriction fragment analysis. Polymorphisms that can be identified
in this manner are termed "restriction fragment length
polymorphisms" ("RFLPs"). RFLPs have been widely used in human and
plant genetic analyses (Glassberg, UK Patent Application 2135774;
Skolnick, et al., Cytogen. Cell Genet. 32:58-67 (1982); Botstein,
et al., Ann. J. Hum. Genet. 32:314-331 (1980); Fischer, et al. (PCT
Application WO90/13668); Uhlen, PCT Application WO90/11369).
[0137] Polymorphisms can also be identified by Single Strand
Conformation Polymorphism (SSCP) analysis. The SSCP technique is a
method capable of identifying most sequence variations in a single
strand of DNA, typically between 150 and 250 nucleotides in length
(Elles, Methods in Molecular Medicine: Molecular Diagnosis of
Genetic Diseases, Humana Press (1996), the entirety of which is
herein incorporated by reference); Orita et al., Genomics 5:874-879
(1989), the entirety of which is herein incorporated by reference).
Under denaturing conditions a single strand of DNA will adopt a
conformation that is uniquely dependent on its sequence
conformation. This conformation usually will be different, even if
only a single base is changed. Most conformations have been
reported to alter the physical configuration or size sufficiently
to be detectable by electrophoresis. A number of protocols have
been described for SSCP including, but not limited to Lee et al.,
Anal. Biochem. 205:289-293 (1992), the entirety of which is herein
incorporated by reference; Suzuki et al., Anal. Biochem. 192:82-84
(1991), the entirety of which is herein incorporated by reference;
Lo et al., Nucleic Acids Research 20:1005-1009 (1992), the entirety
of which is herein incorporated by reference; Sarkar et al.,
Genomics 13:441-443 (1992), the entirety of which is herein
incorporated by reference). It is understood that one or more of
the nucleic acids of the present invention, may be utilized as
markers or probes to detect polymorphisms by SSCP analysis.
[0138] Polymorphisms may also be found using a DNA fingerprinting
technique called amplified fragment length polymorphism (AFLP),
which is based on the selective PCR amplification of restriction
fragments from a total digest of genomic DNA to profile that DNA.
Vos, et al., Nucleic Acids Res. 23:4407-4414 (1995), the entirety
of which is herein incorporated by reference. This method allows
for the specific co-amplification of high numbers of restriction
fragments, which can be visualized by PCR without knowledge of the
nucleic acid sequence.
[0139] AFLP employs basically three steps. Initially, a sample of
genomic DNA is cut with restriction enzymes and oligonucleotide
adapters are ligated to the restriction fragments of the DNA. The
restriction fragments are then amplified using PCR by using the
adapter and restriction sequence as target sites for primer
annealing. The selective amplification is achieved by the use of
primers that extend into the restriction fragments, amplifying only
those fragments in which the primer extensions match the nucleotide
flanking the restriction sites. These amplified fragments are then
visualized on a denaturing polyacrylamide gel.
[0140] AFLP analysis has been performed on Salix (Beismann, et al.,
Mol. Ecol. 6:989-993 (1997), the entirety of which is herein
incorporated by reference); Acinetobacter (Janssen, et al., Int. J.
Syst. Bacteriol 47:1179-1187 (1997), the entirety of which is
herein incorporated by reference), Aeromonas popoffi (Huys, et al.,
Int. J. Syst. Bacteriol. 47:1165-1171 (1997), the entirety of which
is herein incorporated by reference), rice (McCouch, et al., Plant
Mol. Biol. 35:89-99 (1997), the entirety of which is herein
incorporated by reference); Nandi, et al., Mol. Gen. Genet. 255:1-8
(1997); Cho, et al., Genome 39:373-378 (1996), herein incorporated
by reference), barley (Hordeum vulgare) (Simons, et al., Genomics
44:61-70 (1997), the entirety of which is herein incorporated by
reference; Waugh, et al., Mol. Gen. Genet. 255:311-321 (1997), the
entirety of which is herein incorporated by reference; Qi, et al.,
Mol. Gen. Genet. 254:330-336 (1997), the entirety of which is
herein incorporated by reference; Becker, et al., Mol. Gen. Genet.
249:65-73 (1995), the entirety of which is herein incorporated by
reference), potato (Van der Voort, et al., Mol. Gen. Genet.
255:438-447 (1997), the entirety of which is herein incorporated by
reference; Meksem, et al., Mol. Gen. Genet. 249:74-81 (1995), the
entirety of which is herein incorporated by reference),
Phytophthora infestans (Van der Lee, et al., Fungal Genet. Biol.
21:278-291 (1997), the entirety of which is herein incorporated by
reference), Bacillus anthracis (Keim, et al., J. Bacteriol.
179:818-824 (1997)), Astragalus cremnophylax (Travis, et al., Mol.
Ecol. 5:735-745 (1996), the entirety of which is herein
incorporated by reference), Arabidopsis (Cnops, et al., Mol. Gen.
Genet. 253:32-41 (1996), the entirety of which is herein
incorporated by reference), Escherichia coli (Lin, et al., Nucleic
Acids Res. 24:3649-3650 (1996), the entirety of which is herein
incorporated by reference), Aeromonas (Huys, et al., Int. J. Syst.
Bacteriol. 46:572-580 (1996), the entirety of which is herein
incorporated by reference), nematode (Folkertsma, et al., Mol.
Plant. Microbe Interact. 9:47-54 (1996), the entirety of which is
herein incorporated by reference), tomato (Thomas, et al., Plant J.
8:785-794 (1995), the entirety of which is herein incorporated by
reference), and human (Latorra, et al., PCR Methods Appl. 3:351-358
(1994) the entirety of which is herein incorporated by reference).
AFLP analysis has also been used for fingerprinting mRNA (Money, et
al., Nucleic Acids Res. 24:2616-2617 (1996), the entirety of which
is herein incorporated by reference; Bachem, et al., Plant J.
9:745-753 (1996), the entirety of which is herein incorporated by
reference). It is understood that one or more of the nucleic acid
molecules of the present invention, may be utilized as markers or
probes to detect polymorphisms by AFLP analysis for fingerprinting
mRNA.
[0141] Polymorphisms may also be found using random amplified
polymorphic DNA (RAPD) (Williams et al., Nucl. Acids Res.
18:6531-6535 (1990), the entirety of which is herein incorporated
by reference) and cleavable amplified polymorphic sequences (CAPS)
(Lyamichev et al., Science 260:778-783 (1993), the entirety of
which is herein incorporated by reference). It is understood that
one or more of the nucleic acid molecules of the present invention,
may be utilized as markers or probes to detect polymorphisms by
RAPD or CAPS analysis.
[0142] Nucleic acid molecules of the present invention can be used
to monitor expression. A microarray-based method for
high-throughput monitoring of plant gene expression may be utilized
to measure gene-specific hybridization targets. This `chip`-based
approach involves using microarrays of nucleic acid molecules as
gene-specific hybridization targets to quantitatively measure
expression of the corresponding plant genes (Schena et al., Science
270:467-470 (1995), the entirety of which is herein incorporated by
reference; Shalon, Ph.D. Thesis. Stanford University (1996), the
entirety of which is herein incorporated by reference). Every
nucleotide in a large sequence can be queried at the same time.
Hybridization can be used to efficiently analyze nucleotide
sequences.
[0143] Several microarray methods have been described. One method
compares the sequences to be analyzed by hybridization to a set of
oligonucleotides or cDNA molecules representing all possible
subsequences (Bains and Smith, J. Theor. Biol. 135:303 (1989), the
entirety of which is herein incorporated by reference). A second
method hybridizes the sample to an array of oligonucleotide or cDNA
probes. An array consisting of oligonucleotides or cDNA molecules
complementary to subsequences of a target sequence can be used to
determine the identity of a target sequence, measure its amount,
and detect differences between the target and a reference sequence.
Nucleic acid molecule microarrays may also be screened with protein
molecules or fragments thereof to determine nucleic acid molecules
that specifically bind protein molecules or fragments thereof.
[0144] Additionally, microarrays of BACs may be prepared to
sufficiently cover 3.times. of an entire genome. Such microarrays
can be used in a variety of genomics experiments including gene
mapping, DNA fingerprinting and promoter identification.
Microarrays of genomic DNA can also be used for parallel analysis
of genomes at single gene resolution (Lemieux et al., Molecular
Breeding 277-289 (1988), the entirety of which is herein
incorporated by reference). It is understood that one or more of
the molecules of the present invention, preferably one or more of
the nucleic acid molecules or protein molecules or fragments
thereof of the present invention may be utilized in a genomic
microarray based method. In a preferred embodiment of the present
invention, one or more of the Glycine max nucleic acid molecules or
protein molecules or fragments thereof of the present invention may
be utilized in a genomic microarray based method. For example,
Genomic Mismatch Scanning (GMS), a hybridization-based method of
linkage analysis that allows rapid identification of regions of
identity-by-descent between two related individuals, can be carried
out with microarrays. GMS is reported to have been used to identify
genetically common chromosomal segments based on the ability of
these DNA sequences to form extensive regions of mismatch-free
heteroduplexes. A series of enzymatic steps, coupled with filter
binding, is used to selectively remove heteroduplexes that contain
mismatches (i.e., chromosomal regions that do not share identity-by
descent.). Fragments of chromosomal DNA representing inherited
regions are hybridized to a microarray of ordered genomic clones
and positive hybridization signals pinpoint regions of
identity-by-descent at high resolution (Lemieux et al., Molecular
Breeding 277-289 (1988))
[0145] It is understood that one or more of the molecules of the
present invention, preferably one or more of the nucleic acid
molecules or protein molecules or fragments thereof of the present
invention may be utilized in a GMS microarray based method to
locate regions of identity-by-descent between related individuals.
In a preferred embodiment of the present invention, one or more of
the Glycine max nucleic acid molecules or protein molecules or
fragments thereof of the present invention may be utilized in a GMS
microarray based method to locate regions of identity-by-descent
between related individuals. The GMS microarray approach can also
be used as a tool to map mutagenic traits. For example, in yeast,
the entire genomic sequence is known and it has been reported that
the genes responsible for growth at elevated temperature, a trait
required for the pathogenicity of certain yeast strains, may be
determined using GMS (Lemieux et al, Molecular Breeding 277-289
(1988)). By analyzing the inheritance of large numbers of tetrads
derived from crosses of pathogenic and wild type strains, all the
genes responsible for a yeast strain's ability to grow at
42.degree. C., for example, could be identified.
[0146] It is understood that one or more of the molecules of the
present invention, preferably one or more of the nucleic acid
molecules or protein molecules or fragments thereof of the present
invention may be utilized in a GMS microarray based method to map
multigenic traits. In a preferred embodiment of the present
invention, one or more of the Glycine max nucleic acid molecules or
protein molecules or fragments thereof of the present invention may
be utilized in a GMS microarray based method to map multigenic
traits.
[0147] Plant repeat elements may be used with GMS microarraying to
identify species specific chromosomes in another species
background. For example, the maize genome contains moderately
repetitive DNA sequences (ZLRS) representing about 2500 copies per
haploid genome; these sequences are present in the genus Zea and
absent in other graminaceous species. Ananiev et al., (Proc. Natl.
Acad. Sci. (U.S.A.) 94:3526-3529 (1997), all of which are herein
incorporated by reference in their entirety) have reported unusual
plants with individual maize chromosomes added to a complete oat
genome generated by embryo rescue from oat (Avena sativa).times.Zea
mays crosses. By using highly repetitive maize-specific sequences
as probes, Ananiev et al. (1997) were able to selectively isolate
cosmid clones containing maize genomic DNA.
[0148] It is understood that one or more of the molecules of the
present invention, preferably one or more of the nucleic acid
molecules or protein molecules or fragments thereof of the present
invention may be utilized in a GMS microarray based method using
repeat elements to selectively isolate clones containing species
specific DNA. In a preferred embodiment of the present invention,
one or more of the Glycine max nucleic acid molecules or protein
molecules or fragments thereof of the present invention may be
utilized in a GMS microarray based method to selectively isolate
clones containing species specific DNA. A particular preferred
microarray embodiment of the present invention is a microarray
comprising nucleic acid molecules encoding genes that are
homologues of known genes or nucleic acid molecules that comprise
genes or fragments thereof that elicit only limited or no matches
to known genes. A further preferred microarray embodiment of the
present invention is a microarray comprising nucleic acid molecules
encoding genes or fragments thereof that are homologues of known
genes and nucleic acid molecules that comprise genes or fragments
thereof that elicit only limited or no matches to known genes. A
further preferred microarray embodiment of the present invention is
a microarray comprising nucleic acid molecules encoding genes or
fragments thereof that elicit only limited or no matches to known
genes.
[0149] It is understood that one or more of the molecules of the
present invention, preferably one or more of the nucleic acid
molecules or protein molecules or fragments thereof of the present
invention may be utilized in a microarray based method. In a
preferred embodiment of the present invention, one or more of the
Glycine max nucleic acid molecules or protein molecules or
fragments thereof of the present invention may be utilized in a
microarray based method.
[0150] Nucleic acid molecules of the present invention may be used
in site directed mutagenesis. Site-directed mutagenesis may be
utilized to modify nucleic acid sequences, particularly as it is a
technique that allows one or more of the amino acids encoded by a
nucleic acid molecule, to be altered (e.g. a threonine to be
replaced by a methionine). Three basic methods for site-directed
mutagenesis are often employed. These are cassette mutagenesis
(Wells et al., Gene 34:315-23 (1985), the entirety of which is
herein incorporated by reference), primer extension (Gilliam et
al., Gene 12:129-137 (1980), the entirety of which is herein
incorporated by reference); Zoller and Smith, Methods Enzymol.
100:468-500 (1983), the entirety of which is herein incorporated by
reference; and Dalbadie-McFarland et al., Proc. Natl. Acad. Sci.
(U.S.A.) 79:6409-6413 (1982), the entirety of which is herein
incorporated by reference) and methods based upon PCR (Scharf et
al., Science 233:1076-1078 (1986), the entirety of which is herein
incorporated by reference; Higuchi et al., Nucleic Acids Res.
16:7351-7367 (1988), the entirety of which is herein incorporated
by reference).
[0151] Any of the nucleic acid molecules of the present invention
may either be modified by site-directed mutagenesis or used as, for
example, nucleic acid molecules that are used to target other
nucleic acid molecules for modification. It is understood that
mutants with more than one altered nucleotide can be constructed
using techniques that practitioners skilled in the art are familiar
with such as isolating restriction fragments and ligating such
fragments into an expression vector.
[0152] ApBACwich system has been developed to achieve site-directed
integration of DNA into the genome. A 150 kb cotton BAC DNA is
reported to have been transferred into a specific lox site in
tobacco by biolistic bombardment and Cre-lox site specific
recombination.
[0153] A construct or vector comprising a nucleic acid molecules of
the present invention may be used in transformation. Exogenous
genetic material may be transferred into a plant cell and the plant
cell regenerated into a whole, fertile or sterile plant. Exogenous
genetic material is any genetic material, whether naturally
occurring or otherwise, from any source that is capable of being
inserted into any organism. In a preferred embodiment of the
present invention the exogenous genetic material can include
Glycine max genetic material. Such genetic material may be
transferred into either monocotyledons and dicotyledons including
but not limited to the plants, Zea mays and Arabidopsis thaliana
and soybean (See specifically, Chistou, Particle Bombardment for
Genetic Engineering of Plants, pp. 63-69 (Zea mays), ppSO-60
(soybean), Biotechnology Intelligence Unit, Academic Press, San
Diego, Calif. (1996), the entirety of which is herein incorporated
by reference and generally Chistou, Particle Bombardment for
Genetic Engineering of Plants, Biotechnology Intelligence Unit,
Academic Press, San Diego, Calif. (1996), the entirety of which is
herein incorporated by reference).
[0154] Transfer of a nucleic acid that encodes for a protein can
result in overexpression of that protein in a transformed cell or
transgenic plant. One or more of the proteins or fragments thereof
encoded by nucleic acid molecules of the present invention may be
overexpressed in a transformed cell or transformed plant. Such
overexpression may be the result of transient or stable transfer of
the exogenous material.
[0155] Exogenous genetic material may be transferred into a plant
cell by the use of a DNA vector or construct designed for such a
purpose. In a preferred embodiment, the exogenous genetic material
comprises a nucleic acid molecule of the present invention. Vectors
have been engineered for transformation of large DNA inserts into
plant genomes. Vectors have been designed to replicate in both E.
coli and A. tumefaciens and have all of the features required for
transferring large inserts of DNA into plant chromosomes (Choi and
Wing, at the website genome.clemson.edu/protocols2-nj.html July,
1998). ApBACwich system has been developed to achieve site-directed
integration of DNA into the genome. A 150 kb cotton BAC DNA is
reported to have been transferred into a specific lox site in
tobacco by biolistic bombardment and Cre-lox site specific
recombination.
[0156] A construct or vector may include a plant promoter to
express the protein or protein fragment of choice. A number of
promoters which are active in plant cells have been described in
the literature. These include the nopaline synthase (NOS) promoter
(Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749 (1987),
the entirety of which is herein incorporated by reference), the
octopine synthase (OCS) promoter (which are carried on
tumor-inducing plasmids of Agrobacterium tumefaciens), the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987), the
entirety of which is herein incorporated by reference) and the CAMV
35S promoter (Odell et al., Nature 313:810-812 (1985), the entirety
of which is herein incorporated by reference), the figwort mosaic
virus 35S-promoter, the light-inducible promoter from the small
subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the
Adh promoter (Walker et al., Proc. Natl. Acad. Sci. (U.S.A.)
84:6624-6628 (1987), the entirety of which is herein incorporated
by reference), the sucrose synthase promoter (Yang et al., Proc.
Natl. Acad. Sci. (U.S.A.) 87:4144-4148 (1990), the entirety of
which is herein incorporated by reference), the R gene complex
promoter (Chandler et al., The Plant Cell 1:1175-1183 (1989), the
entirety of which is herein incorporated by reference), and the
chlorophyll a/b binding protein gene promoter, etc. These promoters
have been used to create DNA constructs which have been expressed
in plants; see, e.g., PCT publication WO 84/02913, herein
incorporated by reference in its entirety.
[0157] Promoters which are known or are found to cause
transcription of DNA in plant cells can be used in the present
invention. Such promoters may be obtained from a variety of sources
such as plants and plant viruses. It is preferred that the
particular promoter selected should be capable of causing
sufficient expression to result in the production of an effective
amount of protein to cause the desired phenotype. In addition to
promoters which are known to cause transcription of DNA in plant
cells, other promoters may be identified for use in the current
invention by screening a plant cDNA library for genes which are
selectively or preferably expressed in the target tissues or
cells.
[0158] For the purpose of expression in source tissues of the
plant, such as the leaf, seed, root or stem, it is preferred that
the promoters utilized in the present invention have relatively
high expression in these specific tissues. For this purpose, one
may choose from a number of promoters for genes with tissue- or
cell-specific or -enhanced expression. Examples of such promoters
reported in the literature include the chloroplast glutamine
synthetase GS2 promoter from pea (Edwards et al., Proc. Natl. Acad.
Sci. (U.S.A.) 87:3459-3463 (1990), herein incorporated by reference
in its entirety), the chloroplast fructose-1,6-biphosphatase
(FBPase) promoter from wheat (Lloyd et al., Mol. Gen. Genet.
225:209-216 (1991), herein incorporated by reference in its
entirety), the nuclear photosynthetic ST-LS1 promoter from potato
(Stockhaus et al., EMBO J. 8:2445-2451 (1989), herein incorporated
by reference in its entirety), the phenylalanine ammonia-lyase
(PAL) promoter and the chalcone synthase (CHS) promoter from
Arabidopsis thaliana. Also reported to be active in
photosynthetically active tissues are the ribulose-1,5-bisphosphate
carboxylase (RbcS) promoter from eastern larch (Larix laricina),
the promoter for the cab gene, cab6, from pine (Yamamoto et al.,
Plant Cell Physiol. 35:773-778 (1994), herein incorporated by
reference in its entirety), the promoter for the Cab-1 gene from
wheat (Fejes et al., Plant Mol. Biol. 15:921-932 (1990), herein
incorporated by reference in its entirety), the promoter for the
CAB-1 gene from spinach (Lubberstedt et al., Plant Physiol.
104:997-1006 (1994), herein incorporated by reference in its
entirety), the promoter for the cab1R gene from rice (Luan et al.,
Plant Cell. 4:971-981 (1992), the entirety of which is herein
incorporated by reference), the pyruvate, orthophosphate dikinase
(PPDK) promoter from Zea mays (Matsuoka et al., Proc. Natl. Acad.
Sci. (U.S.A.) 90:9586-9590 (1993), herein incorporated by reference
in its entirety), the promoter for the tobacco Lhcb1*2 gene (Cerdan
et al., Plant Mol. Biol. 33:245-255. (1997), herein incorporated by
reference in its entirety), the Arabidopsis thaliana SUC2
sucrose-H+ symporter promoter (Truernit et al., Planta. 196:564-570
(1995), herein incorporated by reference in its entirety), and the
promoter for the thylacoid membrane proteins from spinach (psaD,
psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Other promoters for
the chlorophyll a/b-binding proteins may also be utilized in the
present invention, such as the promoters for LhcB gene and PsbP
gene from white mustard (Sinapis alba; Kretsch et al., Plant Mol.
Biol. 28:219-229 (1995), the entirety of which is herein
incorporated by reference).
[0159] For the purpose of expression in sink tissues of the plant,
such as the tuber of the potato plant, the fruit of tomato, or the
seed of Zea mays, wheat, rice, and barley, it is preferred that the
promoters utilized in the present invention have relatively high
expression in these specific tissues. A number of promoters for
genes with tuber-specific or -enhanced expression are known,
including the class I patatin promoter (Bevan et al., EMBO J.
8:1899-1906 (1986); Jefferson et al., Plant Mol. Biol. 14995-1006
(1990), both of which are herein incorporated by reference in its
entirety), the promoter for the potato tuber ADPGPP genes, both the
large and small subunits, the sucrose synthase promoter (Salanoubat
and Belliard, Gene. 60:47-56 (1987), Salanoubat and Belliard, Gene.
84:181-185 (1989), both of which are incorporated by reference in
their entirety), the promoter for the major tuber proteins
including the 22 kd protein complexes and proteinase inhibitors
(Hannapel, Plant Physiol. 101:703-704 (1993), herein incorporated
by reference in its entirety), the promoter for the granule bound
starch synthase gene (GBSS) (Visser et al., Plant Mol. Biol.
17:691-699 (1991), herein incorporated by reference in its
entirety), and other class I and II patatins promoters
(Koster-Topfer et al., Mol. Gen. Genet. 219:390-396 (1989); Mignery
et al., Gene. 62:27-44 (1988), both of which are herein
incorporated by reference in their entirety).
[0160] Other promoters can also be used to express a fructose 1,6
bisphosphate aldolase gene in specific tissues, such as seeds or
fruits. The promoter for .beta.-conglycinin (Chen et al., Dev.
Genet. 10:112-122 (1989), herein incorporated by reference in its
entirety) or other seed-specific promoters such as the napin and
phaseolin promoters, can be used. The zeins are a group of storage
proteins found in Zea mays endosperm. Genomic clones for zein genes
have been isolated (Pedersen et al., Cell 29:1015-1026 (1982),
herein incorporated by reference in its entirety), and the
promoters from these clones, including the 15 kD, 16 kD, 19 kD, 22
kD, 27 kD, and gamma genes, could also be used. Other promoters
known to function, for example, in Zea mays, include the promoters
for the following genes: waxy, Brittle, Shrunken 2, Branching
enzymes I and II, starch synthases, debranching enzymes, oleosins,
glutelins, and sucrose synthases. A particularly preferred promoter
for Zea mays endosperm expression is the promoter for the glutelin
gene from rice, more particularly the Osgt-1 promoter (Zheng et
al., Mol. Cell. Biol. 13:5829-5842 (1993), herein incorporated by
reference in its entirety). Examples of promoters suitable for
expression in wheat include those promoters for the ADPglucose
pyrophosphorylase (ADPGPP) subunits, the granule bound and other
starch synthases, the branching and debranching enzymes, the
embryogenesis-abundant proteins, the gliadins, and the glutenins.
Examples of such promoters in rice include those promoters for the
ADPGPP subunits, the granule bound and other starch synthases, the
branching enzymes, the debranching enzymes, sucrose synthases, and
the glutelins. A particularly preferred promoter is the promoter
for rice glutelin, Osgt-1. Examples of such promoters for barley
include those for the ADPGPP subunits, the granule bound and other
starch synthases, the branching enzymes, the debranching enzymes,
sucrose synthases, the hordeins, the embryo globulins, and the
aleurone specific proteins.
[0161] Root specific promoters may also be used. An example of such
a promoter is the promoter for the acid chitinase gene (Samac et
al., Plant Mol. Biol. 25:587-596 (1994), the entirety of which is
herein incorporated by reference). Expression in root tissue could
also be accomplished by utilizing the root specific subdomains of
the CaMV35S promoter that have been identified (Lam et al., Proc.
Natl. Acad. Sci. (U.S.A.) 86:7890-7894 (1989), herein incorporated
by reference in its entirety). Other root cell specific promoters
include those reported by Conkling et al. (Conkling et al., Plant
Physiol. 93:1203-1211 (1990), the entirety of which is herein
incorporated by reference).
[0162] Additional promoters that may be utilized are described, for
example, in U.S. Pat. Nos. 5,378,619, 5,391,725, 5,428,147,
5,447,858, 5,608,144, 5,608,144, 5,614,399, 5,633,441, 5,633,435,
and 4,633,436, all of which are herein incorporated in their
entirety. In addition, a tissue specific enhancer may be used
(Fromm et al., The Plant Cell 1:977-984 (1989), the entirety of
which is herein incorporated by reference).
[0163] Constructs or vectors may also include, with the coding
region of interest, a nucleic acid sequence that acts, in whole or
in part, to terminate transcription of that region. For example,
such sequences have been isolated including the Tr7 3' sequence and
the nos 3' sequence (Ingelbrecht et al., The Plant Cell 1:671-680
(1989), the entirety of which is herein incorporated by reference;
Bevan et al., Nucleic Acids Res. 11:369-385 (1983), the entirety of
which is herein incorporated by reference), or the like.
[0164] A vector or construct may also include regulatory elements.
Examples of such include the Adh intron 1 (Callis et al., Genes and
Develop. 1:1183-1200 (1987), the entirety of which is herein
incorporated by reference), the sucrose synthase intron (Vasil et
al., Plant Physiol. 91:1575-1579 (1989), the entirety of which is
herein incorporated by reference) and the TMV omega element (Gallie
et al., The Plant Cell 1:301-311 (1989), the entirety of which is
herein incorporated by reference). These and other regulatory
elements may be included when appropriate.
[0165] A vector or construct may also include a selectable marker.
Selectable markers may also be used to select for plants or plant
cells that contain the exogenous genetic material. Examples of such
include, but are not limited to, a neo gene (Potrykus et al., Mol.
Gen. Genet. 199:183-188 (1985), the entirety of which is herein
incorporated by reference) which codes for kanamycin resistance and
can be selected for using kanamycin, G418, etc.; a bar gene which
codes for bialaphos resistance; a mutant EPSP synthase gene
(Hinchee et al., Bio/Technology 6:915-922 (1988), the entirety of
which is herein incorporated by reference) which encodes glyphosate
resistance; a nitrilase gene which confers resistance to bromoxynil
(Stalker et al., J. Biol. Chem. 263:6310-6314 (1988), the entirety
of which is herein incorporated by reference); a mutant
acetolactate synthase gene (ALS) which confers imidazolinone or
sulphonylurea resistance (European Patent Application 154,204 (Sep.
11, 1985), the entirety of which is herein incorporated by
reference); and a methotrexate resistant DHFR gene (Thillet et al.,
J. Biol. Chem. 263:12500-12508 (1988), the entirety of which is
herein incorporated by reference).
[0166] A vector or construct may also include a transit peptide.
Incorporation of a suitable chloroplast transit peptide may also be
employed (European Patent Application Publication Number 0218571,
the entirety of which is herein incorporated by reference).
Translational enhancers may also be incorporated as part of the
vector DNA. DNA constructs could contain one or more 5'
non-translated leader sequences which may serve to enhance
expression of the gene products from the resulting mRNA
transcripts. Such sequences may be derived from the promoter
selected to express the gene or can be specifically modified to
increase translation of the mRNA. Such regions may also be obtained
from viral RNAs, from suitable eukaryotic genes, or from a
synthetic gene sequence. For a review of optimizing expression of
transgenes, see Koziel et al., Plant Mol. Biol. 32:393-405 (1996),
the entirety of which is herein incorporated by reference.
[0167] A vector or construct may also include a screenable marker.
Screenable markers may be used to monitor expression. Exemplary
screenable markers include a .beta.-glucuronidase or uidA gene
(GUS) which encodes an enzyme for which various chromogenic
substrates are known (Jefferson, Plant Mol. Biol, Rep. 5:387-405
(1987), the entirety of which is herein incorporated by reference;
Jefferson et al., EMBO J. 6:3901-3907 (1987), the entirety of which
is herein incorporated by reference); an R-locus gene, which
encodes a product that regulates the production of anthocyanin
pigments (red color) in plant tissues ((Dellaporta et al., Stadler
Symposium 11:263-282 (1988), the entirety of which is herein
incorporated by reference); a .beta.-lactamase gene (Sutcliffe et
al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741 (1978), the
entirety of which is herein incorporated by reference), a gene
which encodes an enzyme for which various chromogenic substrates
are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase
gene (Ow et al., Science 234:856-859 (1986), the entirety of which
is herein incorporated by reference) a xylE gene (Zukowsky et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 80:1101-1105 (1983), the entirety
of which is herein incorporated by reference) which encodes a
catechol dioxygenase that can convert chromogenic catechols; an
.alpha.-amylase gene (Ikatu et al., Bio/Technol. 8:241-242 (1990),
the entirety of which is herein incorporated by reference); a
tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714
(1983), the entirety of which is herein incorporated by reference)
which encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone which in turn condenses to melanin; an
.alpha.-galactosidase, which will turn a chromogenic
.alpha.-galactose substrate.
[0168] Included within the terms "selectable or screenable marker
genes" are also genes which encode a secretable marker whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers which encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes which can be detected catalytically.
Secretable proteins fall into a number of classes, including small,
diffusible proteins detectable, e.g., by ELISA, small active
enzymes detectable in extracellular solution (e.g.,
.alpha.-amylase, .beta.-lactamase, phosphinothricin transferase),
or proteins which are inserted or trapped in the cell wall (such as
proteins which include a leader sequence such as that found in the
expression unit of extension or tobacco PR-S). Other possible
selectable and/or screenable marker genes will be apparent to those
of skill in the art.
[0169] Methods and compositions for transforming a bacteria and
other microorganisms are known in the art (see for example Sambrook
et al., Molecular Cloning: A Laboratory Manual, Second Edition,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
(1989), the entirety of which is herein incorporated by
reference).
[0170] There are many methods for introducing transforming nucleic
acid molecules into plant cells. Suitable methods are believed to
include virtually any method by which nucleic acid molecules may be
introduced into a cell, such as by Agrobacterium infection or
direct delivery of nucleic acid molecules such as, for example, by
PEG-mediated transformation, by electroporation or by acceleration
of DNA coated particles, etc. (Pottykus, Ann. Rev. Plant Physiol.
Plant Mol. Biol. 42:205-225 (1991), the entirety of which is herein
incorporated by reference; Vasil, Plant Mol. Biol. 25:925-937
(1994), the entirety of which is herein incorporated by reference.
For example, electroporation has been used to transform Zea mays
protoplasts (Fromm et al., Nature 312:791-793 (1986), the entirety
of which is herein incorporated by reference).
[0171] Technology for introduction of DNA into cells is well known
to those of skill in the art. Four general methods for delivering a
gene into cells have been described: (1) chemical methods (Graham
and van der Eb, Virology, 54:536-539 (1973), the entirety of which
is herein incorporated by reference); (2) physical methods such as
microinjection (Capecchi, Cell 22:479-488 (1980), electroporation
(Wong and Neumann, Biochem. Biophys. Res. Commun., 107:584-587
(1982); Fromm et al., Proc. Natl. Acad. Sci. (U.S.A.), 82:5824-5828
(1985); U.S. Pat. No. 5,384,253; and the gene gun (Johnston and
Tang, Methods Cell Biol. 43:353-365 (1994), all of which the
entirety is herein incorporated by reference; (3) viral vectors
(Clapp, Clin. Perinatol., 20:155-168 (1993); Lu et al., J. Exp.
Med., 178:2089-2096 (1993); Eglitis and Anderson, Biotechniques,
6:608-614 (1988), all of which the entirety is herein incorporated
by reference); and (4) receptor-mediated mechanisms (Curiel et al.,
Hum. Gen. Ther., 3:147-154 (1992); Wagner et al., Proc. Natl. Acad.
Sci. U.S.A., 89:6099-6103 (1992), all of which the entirety is
herein incorporated by reference).
[0172] Acceleration methods that may be used include, for example,
microprojectile bombardment and the like. One example of a method
for delivering transforming nucleic acid molecules to plant cells
is microprojectile bombardment. This method has been reviewed by
Yang and Christou, eds., Particle Bombardment Technology for Gene
Transfer, Oxford Press, Oxford, England (1994), the entirety of
which is herein incorporated by reference). Non-biological
particles (microprojectiles) that may be coated with nucleic acids
and delivered into cells by a propelling force. Exemplary particles
include those comprised of tungsten, gold, platinum, and the
like.
[0173] A particular advantage of microprojectile bombardment, in
addition to it being an effective means of reproducibly, and stably
transforming monocotyledons, is that neither the isolation of
protoplasts (Cristou et al., Plant Physiol. 87:671-674 (1988), the
entirety of which is herein incorporated by reference) nor the
susceptibility of Agrobacterium infection is required. An
illustrative embodiment of a method for delivering DNA into maize
cells by acceleration is a biolistics-particle delivery system,
which can be used to propel particles coated with DNA through a
screen, such as a stainless steel or Nytex screen, onto a filter
surface covered with corn cells cultured in suspension. Gordon-Kamm
et al., describes the basic procedure for coating tungsten
particles with DNA (Gordon-Kamm et al., Plant Cell 2:603-618
(1990), the entirety of which is herein incorporated by reference).
The screen disperses the tungsten nucleic acid particles so that
they are not delivered to the recipient cells in large aggregates.
A particle delivery system suitable for use with the present
invention is the helium acceleration PDS-1000/He gun which is
available from Bio-Rad Laboratories (Bio-Rad, Hercules, Calif.)
(Sanford et al., Technique 3:3-16 (1991), the entirety of which is
herein incorporated by reference).
[0174] For the bombardment, cells in suspension may be concentrated
on filters. Filters containing the cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the gun and the cells to be bombarded.
[0175] Alternatively, immature embryos or other target cells may be
arranged on solid culture medium. The cells to be bombarded are
positioned at an appropriate distance below the macroprojectile
stopping plate. If desired, one or more screens are also positioned
between the acceleration device and the cells to be bombarded.
Through the use of techniques set forth herein one may obtain up to
1000 or more foci of cells transiently expressing a marker gene.
The number of cells in a focus which express the exogenous gene
product 48 hours post-bombardment often range from one to ten and
average one to three.
[0176] In bombardment transformation, one may optimize the
prebombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment, and also the nature of the
transforming DNA, such as linearized DNA or intact supercoiled
plasmids. It is believed that pre-bombardment manipulations are
especially important for successful transformation of immature
embryos.
[0177] In another alternative embodiment, plastids can be stably
transformed. Methods disclosed for plastid transformation in higher
plants include particle gun delivery of DNA containing a selectable
marker and targeting of the DNA to the plastid genome through
homologous recombination (Svab et al. Proc. Natl. Acad. Sci.
(U.S.A.) 87:8526-8530 (1990): Svab and Maliga Proc. Natl. Acad.
Sci. (U.S.A.) 90:913-917 (1993)); (Staub, J. M. and Maliga, P. EMBO
J. 12:601-606 (1993), U.S. Pat. Nos. 5,451,513 and 5,545,818 all of
which are herein incorporated by reference in their entirety).
[0178] Accordingly, it is contemplated that one may wish to adjust
various aspects of the bombardment parameters in small scale
studies to fully optimize the conditions. One may particularly wish
to adjust physical parameters such as gap distance, flight
distance, tissue distance, and helium pressure. One may also
minimize the trauma reduction factors by modifying conditions which
influence the physiological state of the recipient cells and which
may therefore influence transformation and integration
efficiencies. For example, the osmotic state, tissue hydration and
the subculture stage or cell cycle of the recipient cells may be
adjusted for optimum transformation. The execution of other routine
adjustments will be known to those of skill in the art in light of
the present disclosure.
[0179] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells because the DNA can
be introduced into whole plant tissues, thereby bypassing the need
for regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example the
methods described (Fraley et al., Biotechnology 3:629-635 (1985);
Rogers et al., Meth. In Enzymol, 153:253-277 (1987), both of which
are herein incorporated by reference in their entirety. Further,
the integration of the Ti-DNA is a relatively precise process
resulting in few rearrangements. The region of DNA to be
transferred is defined by the border sequences, and intervening DNA
is usually inserted into the plant genome as described (Spielmann
et al., Mol. Gen. Genet., 205:34 (1986), the entirety of which is
herein incorporated by reference).
[0180] Modern Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., In: Plant DNA
Infectious Agents, T. Hohn and J. Schell, eds., Springer-Verlag,
New York, pp. 179-203 (1985), the entirety of which is herein
incorporated by reference. Moreover, recent technological advances
in vectors for Agrobacterium-mediated gene transfer have improved
the arrangement of genes and restriction sites in the vectors to
facilitate construction of vectors capable of expressing various
polypeptide coding genes. The vectors described have convenient
multi-linker regions flanked by a promoter and a polyadenylation
site for direct expression of inserted polypeptide coding genes and
are suitable for present purposes (Rogers et al., Meth. In
Enzymol., 153:253-277 (1987), the entirety of which is herein
incorporated by reference). In addition, Agrobacterium containing
both armed and disarmed Ti genes can be used for the
transformations. In those plant strains where
Agrobacterium-mediated transformation is efficient, it is the
method of choice because of the facile and defined nature of the
gene transfer.
[0181] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome. Such
transgenic plants can be referred to as being heterozygous for the
added gene. More preferred is a transgenic plant that is homozygous
for the added structural gene; i.e., a transgenic plant that
contains two added genes, one gene at the same locus on each
chromosome of a chromosome pair. A homozygous transgenic plant can
be obtained by sexually mating (selfing) an independent segregant
transgenic plant that contains a single added gene, germinating
some of the seed produced and analyzing the resulting plants
produced for the gene of interest.
[0182] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating added, exogenous genes. Selfing of
appropriate progeny can produce plants that are homozygous for both
added, exogenous genes that encode a polypeptide of interest.
Back-crossing to a parental plant and out-crossing with a
non-transgenic plant are also contemplated, as is vegetative
propagation.
[0183] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation, and combinations of these
treatments. See for example (Potrykus et al., Mol. Gen. Genet.,
205:193-200 (1986); Lorz et al., Mol. Gen. Genet., 199:178, (1985);
Fromm et al., Nature, 319:791, (1986); Uchimiya et al., Mol. Gen.
Genet.:204:204, (1986); Callis et al., Genes and Development, 1183,
(1987); Marcotte et al., Nature, 335:454, (1988), all of which the
entirety is herein incorporated by reference).
[0184] Application of these systems to different plant strains
depends upon the ability to regenerate that particular plant strain
from protoplasts. Illustrative methods for the regeneration of
cereals from protoplasts are described (Fujimura et al., Plant
Tissue Culture Letters, 2:74, (1985); Toriyama et al., Theor Appl.
Genet. 205:34. (1986); Yamada et al., Plant Cell Rep., 4:85,
(1986); Abdullah et al., Biotechnology, 4:1087, (1986), all of
which the entirety is herein incorporated by reference).
[0185] To transform plant strains that cannot be successfully
regenerated from protoplasts, other ways to introduce DNA into
intact cells or tissues can be utilized. For example, regeneration
of cereals from immature embryos or explants can be effected as
described (Vasil, Biotechnology, 6:397, (1988), the entirety of
which is herein incorporated by reference). In addition, "particle
gun" or high-velocity microprojectile technology can be utilized
(Vasil et al., Bio/Technology 10:667, (1992), the entirety of which
is herein incorporated by reference).
[0186] Using the latter technology, DNA is carried through the cell
wall and into the cytoplasm on the surface of small metal particles
as described (Klein et al., Nature, 328:70, (1987); Klein et al.,
Proc. Natl. Acad. Sci. (U.S.A.), 85:8502-8505, (1988); McCabe et
al., Biotechnology, 6:923, (1988), all of which the entirety is
herein incorporated by reference). The metal particles penetrate
through several layers of cells and thus allow the transformation
of cells within tissue explants.
[0187] Other methods of cell transformation can also be used and
include but are not limited to introduction of DNA into plants by
direct DNA transfer into pollen (Zhou et al., Methods in
Enzymology, 101:433, (1983); Hess et al., Intern Rev. Cytol.,
107:367, (1987); Luo et al., Plant Mol. Biol. Reporter, 6:165,
(1988), all of which the entirety is herein incorporated by
reference), by direct injection of DNA into reproductive organs of
a plant (Pena et al., Nature, 325:274, (1987), the entirety of
which is herein incorporated by reference), or by direct injection
of DNA into the cells of immature embryos followed by the
rehydration of dessicated embryos (Neuhaus et al., Theor. Appl.
Genet., 75:30, (1987), the entirety of which is herein incorporated
by reference).
[0188] The regeneration, development, and cultivation of plants
from single plant protoplast transformants or from various
transformed explants is well known in the art (Weissbach and
Weissbach, In: Methods for Plant Molecular Biology, (Eds.),
Academic Press, Inc., San Diego, Calif., (1988), the entirety of
which is herein incorporated by reference). This regeneration and
growth process typically includes the steps of selection of
transformed cells, culturing those individualized cells through the
usual stages of embryonic development through the rooted plantlet
stage. Transgenic embryos and seeds are similarly regenerated. The
resulting transgenic rooted shoots are thereafter planted in an
appropriate plant growth medium such as soil.
[0189] The development or regeneration of plants containing the
foreign, exogenous gene that encodes a protein of interest is well
known in the art. Preferably, the regenerated plants are
self-pollinated to provide homozygous transgenic plants, as
discussed before. Otherwise, pollen obtained from the regenerated
plants is crossed to seed-grown plants of agronomically important
lines. Conversely, pollen from plants of these important lines is
used to pollinate regenerated plants. A transgenic plant of the
present invention containing a desired polypeptide is cultivated
using methods well known to one skilled in the art.
[0190] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated.
[0191] Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens, and obtaining transgenic plants have
been published for cotton (U.S. Pat. No. 5,004,863, U.S. Pat. No.
5,159,135, U.S. Pat. No. 5,518,908, all of which the entirety is
herein incorporated by reference); soybean (U.S. Pat. No.
5,569,834, U.S. Pat. No. 5,416,011, McCabe et al., Biotechnology
6:923, (1988), Christou et al., Plant Physiol., 87:671-674 (1988),
all of which the entirety is herein incorporated by reference);
Brassica (U.S. Pat. No. 5,463,174, the entirety of which is herein
incorporated by reference); peanut (Cheng et al., Plant Cell Rep.
15:653-657 (1996), McKently et al., Plant Cell Rep. 14:699-703
(1995), all of which the entirety is herein incorporated by
reference); papaya (Yang et al., (1996), the entirety of which is
herein incorporated by reference); pea (Grant et al., Plant Cell
Rep. 15:254-258, (1995), the entirety of which is herein
incorporated by reference).
[0192] Transformation of monocotyledons using electroporation,
particle bombardment, and Agrobacterium have also been reported.
Transformation and plant regeneration have been achieved in
asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (U.S.A.)
84:5345, (1987), the entirety of which is herein incorporated by
reference); barley (Wan and Lemaux, Plant Physiol 104:37, (1994),
the entirety of which is herein incorporated by reference); maize
(Rhodes et al, Science 240:204, (1988), Gordon-Kamm et al., Plant
Cell, 2:603, (1990), Fromm et al., Bio/Technology 8:833, (1990),
Koziel et al., Bio/Technology 11:194, (1993), Armstrong et al.,
Crop Science 35:550-557, (1995), all of which the entirety is
herein incorporated by reference); oat (Somers et al.,
Bio/Technology, 10:1589, (1992), the entirety of which is herein
incorporated by reference); orchardgrass (Horn et al., Plant Cell
Rep. 7:469, (1988), the entirety of which is herein incorporated by
reference); rice (Toriyama et al., Theor Appl. Genet. 205:34,
(1986); Park et al., Plant Mol. Biol., 32:1135-1148, (1996);
Abedinia et al., Aust. J. Plant Physiol. 24:133-141, (1997); Zhang
and Wu, Theor. Appl. Genet. 76:835, (1988); Zhang et al., Plant
Cell Rep. 7:379, (1988); Battraw and Hall, Plant Sci. 86:191-202,
(1992); Christou et al., Bio/Technology 9:957, (1991), all of which
the entirety is herein incorporated by reference); sugarcane (Bower
and Birch, Plant J. 2:409, (1992), the entirety of which is herein
incorporated by reference); tall fescue (Wang et al.,
Bio/Technology 10:691, (1992), the entirety of which is herein
incorporated by reference), and wheat (Vasil et al., Bio/Technology
10:667, (1992), the entirety of which is herein incorporated by
reference; U.S. Pat. No. 5,631,152, the entirety of which is herein
incorporated by reference.
[0193] Assays for gene expression based on the transient expression
of cloned nucleic acid constructs have been developed by
introducing the nucleic acid molecules into plant cells by
polyethylene glycol treatment, electroporation, or particle
bombardment (Marcotte, et al., Nature, 335:454-457 (1988), the
entirety of which is herein incorporated by reference; Marcotte, et
al., Plant Cell, 1:523-532 (1989), the entirety of which is herein
incorporated by reference; McCarty, et al., Cell 66:895-905 (1991),
the entirety of which is herein incorporated by reference; Hattori,
et al., Genes Dev. 6:609-618 (1992), the entirety of which is
herein incorporated by reference; Goff, et al., EMBO J. 9:2517-2522
(1990), the entirety of which is herein incorporated by reference).
Transient expression systems may be used to functionally dissect
gene constructs (See generally, Mailga et al., Methods in Plant
Molecular Biology, Cold Spring Harbor Press (1995)).
[0194] Any of the nucleic acid molecules of the present invention
may be introduced into a plant cell in a permanent or transient
manner in combination with other genetic elements such as vectors,
promoters enhancers etc. Further any of the nucleic acid molecules
of the present invention may be introduced into a plant cell in a
manner that allows for over expression of the protein or fragment
thereof encoded by the nucleic acid molecule.
[0195] Nucleic acid molecules of the present invention may be used
in cosuppression. Cosuppression is the reduction in expression
levels, usually at the level of RNA, of a particular endogenous
gene or gene family by the expression of a homologous sense
construct that is capable of transcribing mRNA of the same
strandedness as the transcript of the endogenous gene (Napoli et
al., Plant Cell 2:279-289 (1990), the entirety of which is herein
incorporated by reference; van der Krol et al., Plant Cell
2:291-299 (1990), the entirety of which is herein incorporated by
reference). Cosuppression may result from stable transformation
with a single copy nucleic acid molecule that is homologous to a
nucleic acid sequence found with the cell (Prolls and Meyer, Plant
J. 2:465-475 (1992), the entirety of which is herein incorporated
by reference) or with multiple copies of a nucleic acid molecule
that is homologous to a nucleic acid sequence found with the cell
(Mittlesten et al., Mol. Gen. Genet. 244: 325-330 (1994), the
entirety of which is herein incorporated by reference). Genes, even
though different, linked to homologous promoters may result in the
cosuppression of the linked genes (Vaucheret, C. R. Acad. Sci. III
316: 1471-1483 (1993), the entirety of which is herein incorporated
by reference).
[0196] This technique has, for example been applied to generate
white flowers from red petunia and tomatoes that do not ripen on
the vine. Up to 50% of petunia transformants that contained a sense
copy of the chalcone synthase (CHS) gene produced white flowers or
floral sectors; this was as a result of the post-transcriptional
loss of mRNA encoding CHS (Flavell, Proc. Natl. Acad. Sci. (U.S.A.)
91:3490-3496 (1994)), the entirety of which is herein incorporated
by reference). Cosuppression may require the coordinate
transcription of the transgene and the endogenous gene, and can be
reset by a developmental control mechanism (Jorgensen, Trends
Biotechnol, 8:340344 (1990), the entirety of which is herein
incorporated by reference; Meins and Kunz, In: Gene Inactivation
and Homologous Recombination in Plants (Paszkowski, J., ed.), pp.
335-348. Kluwer Academic, Netherlands (1994), the entirety of which
is herein incorporated by reference).
[0197] It is understood that one or more of the nucleic acids of
the present invention comprising SEQ ID NO:1 or complement thereof
through SEQ ID NO:36935 or complement thereof, may be introduced
into a plant cell and transcribed using an appropriate promoter
with such transcription resulting in the co-suppression of an
endogenous protein.
[0198] Nucleic acid molecules of the present invention may be used
to reduce gene function. Antisense approaches are a way of
preventing or reducing gene function by targeting the genetic
material (Mol et al., FEBS Lett. 268:427-430 (1990), the entirety
of which is herein incorporated by reference). The objective of the
antisense approach is to use a sequence complementary to the target
gene to block its expression and create a mutant cell line or
organism in which the level of a single chosen protein is
selectively reduced or abolished. Antisense techniques have several
advantages over other `reverse genetic` approaches. The site of
inactivation and its developmental effect can be manipulated by the
choice of promoter for antisense genes or by the timing of external
application or microinjection. Antisense can manipulate its
specificity by selecting either unique regions of the target gene
or regions where it shares homology to other related genes (Hiatt
et al., In Genetic Engineering, Setlow (ed.), Vol. 11, New York:
Plenum 49-63 (1989), the entirety of which is herein incorporated
by reference).
[0199] The principle of regulation by antisense RNA is that RNA
that is complementary to the target mRNA is introduced into cells,
resulting in specific RNA:RNA duplexes being formed by base pairing
between the antisense substrate and the target mRNA (Green et al.,
Annu. Rev. Biochem. 55:569-597 (1986), the entirety of which is
herein incorporated by reference). Under one embodiment, the
process involves the introduction and expression of an antisense
gene sequence. Such a sequence is one in which part or all of the
normal gene sequences are placed under a promoter in inverted
orientation so that the `wrong` or complementary strand is
transcribed into a noncoding antisense RNA that hybridizes with the
target mRNA and interferes with its expression (Takayama and
Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155-184 (1990), the
entirety of which is herein incorporated by reference). An
antisense vector is constructed by standard procedures and
introduced into cells by transformation, transfection,
electroporation, microinjection, or by infection, etc. The type of
transformation and choice of vector will determine whether
expression is transient or stable. The promoter used for the
antisense gene may influence the level, timing, tissue,
specificity, or inducibility of the antisense inhibition.
[0200] It is understood that protein synthesis activity in a plant
cell may be reduced or depressed by growing a transformed plant
cell containing a nucleic acid molecule of the present
invention.
[0201] Antibodies have been expressed in plants (Hiatt et al.,
Nature 342:76-78 (1989), the entirety of which is herein
incorporated by reference; Conrad and Fielder, Plant Mol. Biol.
26:1023-1030 (1994), the entirety of which is herein incorporated
by reference). Cytoplasmic expression of a scFv (single-chain Fv
antibodies) has been reported to delay infection by artichoke
mottled crinkle virus. Transgenic plants that express antibodies
directed against endogenous proteins may exhibit a physiological
effect (Philips et al., EMBO J. 16:4489-4496 (1997), the entirety
of which is herein incorporated by reference; Marion-Poll, Trends
in Plant Science 2:447-448 (1997), the entirety of which is herein
incorporated by reference). For example, expressed anti-abscisic
antibodies reportedly result in a general perturbation of seed
development (Philips et al., EMBO J. 16:4489-4496 (1997)).
[0202] Nucleic acid molecules of the present invention may be used
as antibodies. Antibodies that are catalytic may also be expressed
in plants (abzymes). The principle behind abzymes is that since
antibodies may be raised against many molecules, this recognition
ability can be directed toward generating antibodies that bind
transition states to force a chemical reaction forward (Persidas,
Nature Biotechnology 15:1313-1315 (1997), the entirety of which is
herein incorporated by reference; Baca et al., Ann. Rev. Biophys.
Biomol. Struct. 26:461-493 (1997), the entirety of which is herein
incorporated by reference). The catalytic abilities of abzymes may
be enhanced by site directed mutagenesis. Examples of abzymes are,
for example, set forth in U.S. Pat. No. 5,658,753; U.S. Pat. No.
5,632,990; U.S. Pat. No. 5,631,137; U.S. Pat. No. 5,602,015; U.S.
Pat. No. 5,559,538; U.S. Pat. No. 5,576,174; U.S. Pat. No.
5,500,358; U.S. Pat. No. 5,318,897; U.S. Pat. No. 5,298,409; U.S.
Pat. No. 5,258,289 and U.S. Pat. No. 5,194,585, all of which are
herein incorporated in their entirety.
[0203] It is understood that any of the antibodies of the present
invention may be expressed in plants and that such expression can
result in a physiological effect. It is also understood that any of
the expressed antibodies may be catalytic.
[0204] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant organisms and the
screening and isolating of clones, (see for example, Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press (1989); Mailga et al., Methods in Plant Molecular Biology,
Cold Spring Harbor Press (1995), the entirety of which is herein
incorporated by reference; Birren et al., Genome Analysis:
Analyzing DNA, 1, Cold Spring Harbor, N.Y., the entirety of which
is herein incorporated by reference).
[0205] Computer Media
[0206] One or more of the nucleotide sequence provided in SEQ ID
NO: 1 through SEQ ID NO: 36935 or complements thereof or fragments
of either can be "provided" in a variety of media to facilitate
use. Such a medium can also provide a subset thereof in a form that
allows a skilled artisan to examine the sequences be recorded on
computer readable media. As used herein, "computer readable media"
refers to any medium that can be read and accessed directly by a
computer. Such media include, but are not limited to: magnetic
storage media, such as floppy discs, hard disc, storage medium, and
magnetic tape: optical storage media such as CD-ROM; electrical
storage media such as RAM and ROM; and hybrids of these categories
such as magnetic/optical storage media. A skilled artisan can
readily appreciate how any of the presently known computer readable
mediums can be used to create a manufacture comprising computer
readable medium having recorded thereon a nucleotide sequence of
the present invention.
[0207] As used herein, "recorded" refers to a process for storing
information on computer readable medium. A skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate media
comprising the nucleotide sequence information of the present
invention. A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
skilled artisan can readily adapt any number of data processor
structuring formats (e.g., text file or database) in order to
obtain computer readable medium having recorded thereon the
nucleotide sequence information of the present invention.
[0208] By providing one or more of nucleotide sequences of the
present invention, a skilled artisan can routinely access the
sequence information for a variety of purposes. Computer software
is publicly available which allows a skilled artisan to access
sequence information provided in a computer readable medium. The
examples which follow demonstrate how software which implements the
BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE
(Brutlag et al., Comp. Chem. 17:203-207 (1993), the entirety of
which is herein incorporated by reference) search algorithms on a
Sybase system can be used to identify open reading frames (ORFs)
within the genome that contain homology to ORFs or proteins from
other organisms. Such ORFs are protein-encoding fragments within
the sequences of the present invention and are useful in producing
commercially important proteins such as enzymes used in amino acid
biosynthesis, metabolism, transcription, translation, RNA
processing, nucleic acid and a protein degradation, protein
modification, and DNA replication, restriction, modification,
recombination, and repair.
[0209] The present invention further provides systems, particularly
computer-based systems, which contain the sequence information
described herein. Such systems are designed to identify
commercially important fragments of the nucleic acid molecule of
the present invention. As used herein, "a computer-based system"
refers to the hardware means, software means, and data storage
means used to analyze the nucleotide sequence information of the
present invention. The minimum hardware means of the computer-based
systems of the present invention comprises a central processing
unit (CPU), input means, output means, and data storage means. A
skilled artisan can readily appreciate that any one of the
currently available computer-based system are suitable for use in
the present invention.
[0210] As indicated above, the computer-based systems of the
present invention comprise a data storage means having stored
therein a nucleotide sequence of the present invention and the
necessary hardware means and software means for supporting and
implementing a search means. As used herein, "data storage means"
refers to memory that can store nucleotide sequence information of
the present invention, or a memory access means which can access
manufactures having recorded thereon the nucleotide sequence
information of the present invention. As used herein, "search
means" refers to one or more programs which are implemented on the
computer-based system to compare a target sequence or target
structural motif with the sequence information stored within the
data storage means. Search means are used to identify fragments or
regions of the sequence of the present invention that match a
particular target sequence or target motif. A variety of known
algorithms are disclosed publicly and a variety of commercially
available software for conducting search means are available and
can be used in the computer-based systems of the present invention.
Examples of such software include, but are not limited to,
MacPattern (EMBL), BLASTIN and BLASTIX (NCBIA). One of the
available algorithms or implementing software packages for
conducting homology searches can be adapted for use in the present
computer-based systems.
[0211] The most preferred sequence length of a target sequence is
from about 10 to 100 amino acids or from about 30 to 300 nucleotide
residues. However, it is well recognized that during searches for
commercially important fragments of the nucleic acid molecules of
the present invention, such as sequence fragments involved in gene
expression and protein processing, may be of shorter length.
[0212] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequence(s) are chosen based on a
three-dimensional configuration which is formed upon the folding of
the target motif. There are a variety of target motifs known in the
art. Protein target motifs include, but are not limited to,
enzymatic active sites and signal sequences. Nucleic acid target
motifs include, but are not limited to, promoter sequences, cis
elements, hairpin structures and inducible expression elements
(protein binding sequences).
[0213] Thus, the present invention further provides an input means
for receiving a target sequence, a data storage means for storing
the target sequences of the present invention sequence identified
using a search means as described above, and an output means for
outputting the identified homologous sequences. A variety of
structural formats for the input and output means can be used to
input and output information in the computer-based systems of the
present invention. A preferred format for an output means ranks
fragments of the sequence of the present invention by varying
degrees of homology to the target sequence or target motif. Such
presentation provides a skilled artisan with a ranking of sequences
which contain various amounts of the target sequence or target
motif and identifies the degree of homology contained in the
identified fragment.
[0214] A variety of comparing means can be used to compare a target
sequence or target motif with the data storage means to identify
sequence fragments sequence of the present invention. For example,
implementing software which implement the BLAST and BLAZE
algorithms (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) can
be used to identify open frames within the nucleic acid molecules
of the present invention. A skilled artisan can readily recognize
that any one of the publicly available homology search programs can
be used as the search means for the computer-based systems of the
present invention.
[0215] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE 1
[0216] BACs are stable, non-chimeric cloning systems having genomic
fragment inserts (100-300 kb) and their DNA can be prepared for
most types of experiments including DNA sequencing. BAC vector,
pBeloBAC11, is derived from the endogenous E. coli F-factor
plasmid, which contains genes for strict copy number control and
unidirectional origin of DNA replication. Additionally, pBeloBAC11
has three unique restriction enzyme sites (Hind III, Bam HI and Sph
I) located within the LacZ gene which can be used as cloning sites
for megabase-size plant DNA. Indigo, another BAC vector contains
Hind III and Eco RI cloning sites. This vector also contains a
random mutation in the LacZ gene that allows for darker blue
colonies.
[0217] As an alternative, the P1-derived artificial chromosome
(PAC) can be used as a large DNA fragment cloning vector (Ioannou,
et al., Nature Genet. 6:84-89 (1994), the entirety of which is
herein incorporated by reference; Suzuki, et al., Gene 199:133-137
(1997), the entirety of which is herein incorporated by reference).
The PAC vector has most of the features of the BAC system, but also
contains some of the elements of the bacteriophage P1 cloning
system.
[0218] BAC libraries are generated by ligating size-selected
restriction digested DNA with pBeloBAC11 followed by
electroporation into E. coli. BAC library construction and
characterization is extremely efficient when compared to YAC (yeast
artificial chromosome) library construction and analysis,
particularly because of the chimerism associated with YACs and
difficulties associated with extracting YAC DNA.
[0219] There are two general methods for preparing megabase-size
DNA from plants. The protoplast method yields megabase-size DNA of
high quality with minimal breakage. The process involves preparing
young leaves which are manually feathered with a razor-blade before
being incubated for four to five hours with cell-wall-degrading
enzymes. The second method developed by Zhange et al., Plant J.
7:175-184 (1995) the entirety of which is herein incorporated by
reference is a universal nuclei method that works well for several
divergent plant taxa. Fresh or frozen tissue is homogenized with a
blender or mortar and pestle. Nuclei are then isolated and
embedded. DNA is prepared by the nucleic method often more
concentrated and is reported to contain lower amounts of
chloroplast DNA than the protoplast method.
[0220] Once protoplasts or nuclei are produced, they are embedded
in an agarose matrix as plugs or microbeads. The agarose provides a
support matrix to prevent shearing of the DNA while allowing
enzymes and buffers to diffuse into the DNA. The DNA is purified
and manipulated in the agarose and is stable for more than one year
at 4.degree. C.
[0221] Once high molecular weight DNA has been prepared, it is
fragmented to the desired size range. In general, DNA fragmentation
utilizes two general approaches, 1) physical shearing and 2)
partial digestion with a restriction enzyme that cuts relatively
frequently within the genome. Since physical shearing is not
dependent upon the frequency and distribution of particular
restriction enzymes sites, this method should yield the most random
distribution of DNA fragments. However, the ends of the sheared DNA
fragments must be repaired and cloned directly or restriction
enzyme sites added by the addition of synthetic linkers. Because of
the subsequent steps required to clone DNA fragmented by shearing,
most protocols fragment DNA by partial restriction enzyme
digestion. The advantage of partial restriction enzyme digestion is
that no further enzymatic modification of the ends of the
restriction fragments are necessary. Four common techniques that
can be used to achieve reproducible partial digestion of
megabase-size DNA are 1) varying the concentration of the
restriction enzyme, 2) varying the time of incubation with the
restriction enzyme 3) varying the concentration of an enzyme
cofactor (e.g., Mg.sup.2+) and 4) varying the ratio of endonuclease
to methylase.
[0222] There are three cloning sites in pBeloBAC11, but only Hind
III and Bam HI produce 5' overhangs for easy vector
dephosphorylation. These two restriction enzymes are primarily used
to construct BAC libraries. The optimal partial digestion
conditions for megabase-size DNA are determined by wide and narrow
window digestions. To optimize the optimum amount of Hind III, 1,
2, 3, 10, and 5-units of enzyme are each added to 50 ml aliquots of
microbeads and incubated at 37.degree. C. for 20 minutes
[0223] After partial digestion of megabase-size DNA, the DNA is run
on a pulsed-field gel, and DNA in a size range of 100-500 kb is
excised from the gel. This DNA is ligated to the BAC vector or
subjected to a second size selection on a pulsed field gel under
different running conditions. Studies have previously reported that
two rounds of size selection can eliminate small DNA fragments
co-migrating with the selected range in the first pulse-field
fractionation. Such a strategy results in an increase in insert
sizes and a more uniform insert size distribution. A practical
approach to performing size selections is to first test for the
number of clones/microliter of ligation and insert size from the
first size selected material. If the numbers are good (500 to 2000
white colony/microliter of ligation) and the size range is also,
good (50 to 300 kb) then a second size selection is practical. When
performing a second size selection one expects a 80 to 95% decrease
in the number of recombinant clones per transformation.
[0224] Twenty to two hundred nanograms of the size-selected DNA is
ligated to dephosphorylated BAC vector (molar ratio of 10 to 1 in
BAC vector excess). Most BAC libraries use a molar ratio of 5 to
15:1 (size selected DNA:BAC vector).
[0225] Transformation is carried out by electroporation and the
transformation efficiency for BACs is about 40 to 1,500
transformants from one microliter of ligation product or 20 to 1000
transformants/ng DNA.
[0226] Several tests can be carried out to determine the quality of
a BAC library. Three basic tests to evaluate the quality include:
the genome coverage of a BAC library-average insert size, average
number of clones hybridizing with single copy probes and
chloroplast DNA content.
[0227] The determination of the average insert size of the library
is assessed in two ways. First, during library construction every
ligation is tested to determine the average insert size by assaying
20-50 BAC clones per ligation. DNA is isolated from recombinant
clones using a standard mini preparation protocol, digested with
Not I to free the insert from the BAC vector and then sized using
pulsed field gel electrophoresis (Maule, Molecular Biotechnology
9:107-126 (1998), the entirety of which is herein incorporated by
reference).
[0228] To determine the genome coverage of the library, it is
screened with single copy RFLP markers distributed randomly across
the genome by hybridization. Microtiter plates containing BAC
clones are spotted onto Hybond membranes. Bacteria from 48 or 72
plates are spotted twice onto one membrane resulting in 18,000 to
27,648 unique clones on each membrane in either a 4.times.4 or
5.times.5 orientation. Since each clone is present twice, false
positives are easily eliminated and true positives are easily
recognized and identified.
[0229] Finally, the chloroplast DNA content in the BAC library is
estimated by hybridizing three chloroplast genes spaced evenly
across the chloroplast genome to the library on high density
hybridization filters.
[0230] There are strategies for isolating rare sequences within the
genome. For example, higher plant genomes can range in size from
100 Mb/1C (Arabidopsis) to 15,966 Mb/C (Triticum aestivum),
(Arumuganathan and Earle, Plant Mol Bio Rep. 9:208-219 (1991), the
entirety of which is herein incorporated by reference). The number
of clones required to achieve a given probability that any DNA
sequence will be represented in a genomic library is
N=(ln(1-P))/(ln(1-L/G)) where N is the number of clones required, P
is the probability desired to get the target sequence, L is the
length of the average clone insert in base pairs and G is the
haploid genome length in base pairs (Clarke et al., Cell 9:91-100
(1976) the entirety of which is herein incorporated by
reference).
[0231] The soybean BAC library of the present invention is
constructed in the pBeloBAC11 or similar vector. Inserts are
generated by partial Eco RI or other enzymatic digestion of DNA
from the cultivar A3244. The library provides approximately twenty
fold coverage of the soybean genome.
EXAMPLE 2
[0232] Two basic methods can be used for DNA sequencing, the chain
termination method of Sanger et al., Proc. Natl. Acad. Sci.
(U.S.A.) 74:5463-5467 (1977), the entirety of which is herein
incorporated by reference and the chemical degradation method of
Maxam and Gilbert, Proc. Natl. Acad. Sci. (U.S.A.) 74:560-564
(1977), the entirety of which is herein incorporated by reference.
Automation and advances in technology such as the replacement of
radioisotopes with fluorescence-based sequencing have reduced the
effort required to sequence DNA (Craxton, Methods, 2:20-26 (1991),
the entirety of which is herein incorporated by reference; Ju et
al., Proc. Natl. Acad. Sci. (U.S.A.) 92:4347-4351 (1995), the
entirety of which is herein incorporated by reference; Tabor and
Richardson, Proc. Natl. Acad. Sci. (U.S.A.) 92:6339-6343 (1995),
the entirety of which is herein incorporated by reference).
Automated sequencers are available from, for example, Pharmacia
Biotech, Inc., Piscataway, N.J. (Pharmacia ALF), LI-COR, Inc.,
Lincoln, Nebr. (LI-COR 4,000) and Millipore, Bedford, Mass.
(Millipore BaseStation).
[0233] In addition, advances in capillary gel electrophoresis have
also reduced the effort required to sequence DNA and such advances
provide a rapid high resolution approach for sequencing DNA samples
(Swerdlow and Gesteland, Nucleic Acids Res. 18:1415-1419 (1990);
Smith, Nature 349:812-813 (1991); Luckey et al., Methods Enzymol.
218:154-172 (1993); Lu et al., J. Chromatog. A. 680:497-501 (1994);
Carson et al., Anal. Chem. 65:3219-3226 (1993); Huang et al., Anal.
Chem. 64:2149-2154 (1992); Kheterpal et al., Electrophoresis
17.1852-1859 (1996); Quesada and Zhang, Electrophoresis
17:1841-1851 (1996); Baba, Yakugaku Zasshi 117:265-281 (1997), all
of which are herein incorporated by reference in their
entirety).
[0234] A number of sequencing techniques are known in the art,
including fluorescence-based sequencing methodologies. These
methods have the detection, automation and instrumentation
capability necessary for the analysis of large volumes of sequence
data. Currently, the 377 DNA Sequencer (Perkin-Elmer Corp., Applied
Biosystems Div., Foster City, Calif.) allows the most rapid
electrophoresis and data collection. With these types of automated
systems, fluorescent dye-labeled sequence reaction products are
detected and data entered directly into the computer, producing a
chromatogram that is subsequently viewed, stored, and analyzed
using the corresponding software programs. These methods are known
to those of skill in the art and have been described and reviewed
(Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring
Harbor, N.Y., the entirety of which is herein incorporated by
reference).
EXAMPLE 3
[0235] To identify sequences containing microsatellites or simple
sequence repeats (SSR), a SSR repeat pattern library is generated
by using a Perl program, SSR_generator.pl, developed at Monsanto.
The library contains repeat patterns of di-, tri-, tetra-, penta-
and hexa-nucleotide repeats, a total of 5421 patterns. The length
of di-, tri-, tetra-, penta- and hexa-nucleotide repeat units were
18, 12, 9, 5 and 4, respectively. These repeat patterns are used to
search against the BAC-end sequence databases by the BLASTN
program. If the search is performed on both strands, complementary
and replicated patterns of an SSR library are removed from the
library to avoid redundancy of SSRs. For di-nucleotide repeats,
there are four unique patterns, i.e. (CA)n, (CT)n, (CG)n and (AT)n.
Product scores are used as a criteria to extract potential SSRs
from BAC-ends. If a product score is equal or greater than 90, the
sequences are further examined.
[0236] The SSR-containing sequences identified from BAC ends are
searched against each other as well as the existing SSR collections
by using BLASTN, and clustering of the sequences is performed by
using CLUSTER2, a tool developed at Monsanto. The minimal
match-length is set to 100 base pairs. Any redundant sequences are
removed and the unique ones are then passed through a visible
inspection to further remove those with not enough flanking
sequences for primer design and those with substantial ambiguous
nucleotides.
[0237] Primers are designed from good quality unique sequences. A
public available primer design software program, PRIMER 3,
(Cambridge, Mass.) is used. PRIMER 3 can be accessed though the
internet at (on the Worldwide web at
genome.wi.mit.edu/cgi-bin/primer/primer3.cgi). Default parameters
are used except those for product size and primer size are changed.
Product Size is Min: 80, Opt: 100, Max: 120, while Primer Size is
Min: 18, Opt: 22 and Max: 27. Oligos are synthesized by Genosis
Biotechnologies, Inc (Houston, Tex.).
[0238] The above protocols are used to develop primers from
Sequence id GM_M02_A2_B07_MR_MR containing the following nucleotide
composition: TABLE-US-00001
AGGCGTTTTNCCTTGATACCTTCGNAGGTCCANCCTTTTNCTTGCTGTAT
CGACTCATTAACACCAAGCTCGGTGAGCACTCTGAAGATTATGACAACTT
TCGNTGATCTTTTTGTCATCGATATTNTAGNAGAGACCAATCTTTCTTCT
TCAAATGTCGCTCATGATATTTATTGTAATTATCTTCAATGTATGTCCAA
AAAGTTAACCTTTTTTGGACCCCCACAATAGAAATCTTTGAAATATTTAG
CCATGTGTTGGCAAGCCATTCATATTTCTTTGCGGAGAAACATGATCTAT
TGTGTCTTTCGGATGCTTCTTCTATGTcttcttcttcttcttcttcttct
tcttcttcttCATTGACCACAATATTATCCAACTCAACTTAGGTGCAAAA
TGGTGGAATTTGAGACTTTGACGCANAGTCAGATGGTGCGTCATGCTCTT
TCATTACATTGGACATCATNTACTACCCTTTGAAGACCCTCGATCCATGG
AAGGGTTAATTGGTG
[0239] This sequence contains CTT dinucleotide repeats with a
repeat unit of 11. Using the Primer 3 program, two primers are
selected: SER157F GTGTCTTTCGGATGCTTCTTCT and SER157R
CACCATTTTGCACCTAAGTTGA. When these two primers are used to amplify
genomic DNAs from eight different varieties, Minsoy, Noir, PIC,
HS-1, A3244, H6686, A0868 and H5088, three alleles are detected.
Sizes of these alleles ranged from 80 to 110 bp The size variation
in the PCR products result from repeat numbers in different
varieties.
PCR Reaction Conditions
[0240] Genomic DNA is isolated from young leaves of Glycine max or
Glycine soja plants. Two leaf discs are collected (approximately 40
mg) from a healthy leaf and stored on wet ice or at 4.degree. C.
Tissue samples are then freeze-dried and stored at -20.degree. C.
or -80.degree. C. The frozen samples are kept as dry as possible
and sealed from contact with the atmosphere. The freeze-dried
samples from -20.degree. C. or -80.degree. C., are allowed to warm
up to room temperature prior to unsealing or opening. One leaflet
(or 2 leaf discs) is inserted into an 1.5 ml Eppendorf tube, placed
on dry ice, and crushed with a wooden dowel. Approximately 200
.mu.l of microprep buffer (25 ml extraction buffer (350 mM
sorbitol, 100 mM Tris-base, 5 mM EDTA-Na.sub.2), 25 ml nuclei lysis
buffer (1M Tris/HCl, 0.5 M EDTA, 5 M NaCl, 2% CTAB), 10 ml 5%
sarkosyl, 0.1 g Na bisulfite) is added to each sample. The sample
is then homogenized. An additional 55011 of microprep buffer is
added, vortexed for about 30-60 seconds, and incubated at
65.degree. C. for about 60 minutes. About 700 .mu.l
chloroform/isoamyl (24:1) is added, mixed well for about 10-30
seconds. Centrifugation of the tubes is performed at approximately
10,000 rpm for 5 minutes in a microcentrifuge. The aqueous phase is
transferred into a new tube and RNA is removed from the extract by
the addition of 30 .mu.l of RNase (10 mg/ml) to the aqueous phase
and incubated for 1 hour at room temperature. Approximately 500
.mu.l ice-cold isopropanol is added to the aqueous extract, and the
tubes inverted until the DNA precipitated. The precipitated
solution is kept at 4.degree. C. for about 1 hour or overnight.
Centrifugation of the tubes is performed at approximately 10,000
rpm for 5 minutes in a microcentrifuge. The supernatant is
discarded and the pellet washed 1-3 times with 200 .mu.l 70%
ethanol. The ethanol is removed using a micropipette and pellet
dried at 37.degree. C. for 10 minutes. The DNA is dissolved in 50
.mu.l TE (10 mM Tris-HCl pH8.0, 0.1 mM EDTA), then kept overnight
at 4.degree. C. Centrifugation of the tubes is performed at
approximately 10,000 rpm for 5 minutes and then the supernatant is
transferred into new tubes. Using this method, approximately 2
.mu.g of DNA per mg of fresh leaf tissue is extracted.
[0241] DNA concentration is measured by a Spectrometry (Molecular
Devices, Sunnyvale, Calif.) and adjusted to proper concentration
for use as template. The total volume for PCR reaction is 20 ul.
The reaction mixture contains: Template DNA at a concentration of
15 ng, 0.15 uM of primer, 0.03 unit of Taq DNA polymerase (Perkin
Elmer), 50 uM of dNTP, the Reaction buffer contains, 10 mM Tris.HCl
pH8.5, 1.5 mM MgCl2, 50 mM KCl and water is added to a total volume
of 20 ul.
[0242] The PCR is performed on a Perkin Elmer DNA Thermal Cycler
9700 using the following cycle profile: hold at 94.degree. C. for 3
min, 32 cycles of 94.degree. C. for 25 second, 47.degree. C. for 25
second and 72.degree. C. for 25 second, and 72.degree. C. for 3 min
of final extension.
[0243] An acrylamide gel is prepared using 56.5 ml water, 3.5 ml
10.times. TAE buffer, 10.5 ml 40 acrylamide stock solution, 50
.mu.l TEMED, 0.06 g ammonium persulfate. To each PCR, sample 20
.mu.l of formamide loading dye is added to each sample and the
samples are denatured at 90.degree. C. for 3 minutes with a
4.degree. C. hold in a thermocycler. 1.5 .mu.l of each sample is
loaded onto the gel. Gels are run at constant wattage to give a
constant heat development during electrophoresis at 40 to 50
Volt/cm of the gel length. Gels should be run at approximately
50.degree. C. during electrophoresis. Electrophoresis is stopped
when the Bromophenol blue dye is at the bottom of the gel. After
electrophoresis, the gel is stained in 1.times.SYBR solution for 15
to 20 minutes with vigorous shaking. A Gel image is recorded using
an Alpha-InnoTech imager.
EXAMPLE 4
[0244] In order to create a file containing complex repeats, the
GCG (Madison, Wis.) REPEAT program is used to determine initial
internal repeats. Stringency is defined as 19 matched bases out of
every contiguous 20 bases in the repeated diagonals part of the
REPEAT program algorithm. After the REPEAT program is run on the
STCs, a REPEAT output filed is processed with the UNIX utilities
grep, sort, uniq and sed to produce a GCG pattern file. The GCG
pattern file is broken into size groups: <20, 20-39, 40-59,
60-79, 80-89, 100-119, 120-139, 140-159, 160-179, 180-199, 200-219,
and >220. Each pattern group is compared against the entire STC
library or subset thereof using the GCG FINDPATTERNS program.
Sequences of size 1-19 are allowed no mismatches. The 20-39 group
are allowed one mismatch. A pattern of size n is allowed floor
(n/20) mismatches. Patterns that occurred in at least 100 STCs are
selected in this step. The results of the FINDPATTERNS program is
post-processed with the UNIX utilities grep, sort and uniq and with
the GCG REFORMAT program to produce GCG sequence files. Each
sequence file is derived from a selected pattern and placed in a
subdirectory that corresponds to its size group. GELSTART,
GELENTER, GELMERGE and GELASSEMBLE are used to coalesce similar
sequences of each size group. Patterns are 90% similar before they
are aligned and the patterns overlap by at least two thirds of the
modal length in their length group. The GELSTART program creates a
subdirectory which contains the individual and the coalesced
consensus sequences. The consensus sequences are placed into a
single directory and a FASTA style sequence library is constructed
from it. The REPEAT-MASKER program is used to mask the original
STCs. The unmasked sequences that remain afterward are concatenated
into 100 KB pseudo-sequences. The pseudo-sequences are fed back
into this algorithm and the new repeat patterns that result are
added to the repeat library. The algorithm is iterated 3 times.
[0245] The repeat library is compared to the STCs using NCBI BLASTN
version 2.0. HSPs are reported if they satisfied the criteria
of:
[0246] "observed fractional match">="allowed fractional
match"
[0247] where "observed fractional match" is defined as:
[0248] "fraction of HSP similarity".times."fraction of query
sequence in HSP"
[0249] and "allowed fractional match" is defined as:
[0250] ("repeat length"-"floor (repeat length/20)")/"repeat
length"
[0251] Alternatively, the repeat library is compared to the STCs by
an algorithm that is written in the C programming language and is
compiled with optimization. Using a repeat library patterns file
containing 3,302 complex repeat sequences from Glycine max, 306,271
Glycine max STC sequences are searched. 1,599,791 repeat
coordinates are identified in these sequences.
[0252] STC and repeat library DNA sequences are represented by the
characters A, C, G, and T. Ambiguous sequence characters allow for
combinations of these characters, as defined by the IUPAC-IUB (the
Wisconsin Package version 10.0, Genetics Computer Group, Madison,
Wis.). For example, A or T is represented by W, G or C by S, and A
or C or G or T by N. DNA sequence characters are represented as 4
binary digits (bits), where 0001 represents A, 0010 represents C,
0100 represents G, and 1000 represents T. Using standard Boolean
logic, A or T (W) is equivalent to applying the logical OR operator
to 0001 and 1000, the result being 1001. The table below shows all
standard symbols and their computer representation for this method.
TABLE-US-00002 IUPAC-IUB Computer Symbol Meaning Representation A A
0001 C C 0010 G G 0100 T T 1000 K G or T (Keto) 1100 M A or C
(aMino) 0011 R A or G (puRine) 0101 S G or C (Strong pairing) 0110
W A or T (Weak pairing) 1001 Y C or T (pYrimidine) 1010 B C or G or
T (not A) 1110 D A or G or T (not C) 1101 H A or C or T (not G)
1011 V A or C or G (not T or U) 0111 N A or C or G or T 1111
When matching sequence patterns, a match occurs only when a symbol
in the sequence being searched is a subset of the symbol appearing
in the pattern. For example, an A in the pattern will match only an
A in the sequence, whereas an R in the pattern will match any of A,
G, or R (but no other symbols). The AND operator is applied to the
computer representation of the pattern symbol and the sequence
symbol, and a match occurs if the result is identical to the
sequence symbol. For example, A matches A because 0001 (pattern)
AND 0001 (sequence) equals 0001 (result), and the result equals the
sequence. An R in the pattern matches an A in the sequence because
0101 (pattern) AND 0001 (sequence) equals 0001 (result), and the
result equals the sequence. An S in the pattern does not match an A
in the sequence: 0110 (pattern) AND 0001 (sequence) equals 0000
(result), the result not matching the sequence. Using this
algorithm, pattern matching becomes a byte by byte comparison using
the AND operator.
[0253] The algorithm allows the user to define the number of
mismatches as a fraction of the number of characters in the
pattern. For example, a 5% mismatch frequency allows for one
mismatch every 20 pattern characters. This works out to 0
mismatches for a pattern of 1-19 characters, 1 mismatch for a
pattern of 20-39 characters, 2 mismatches for a pattern of 40-59
characters, and so on.
[0254] The searching algorithm aligns the pattern sequence with the
DNA sequence at every possible position on both DNA strands and
counts the number of mismatches in the alignment. If the number of
mismatches is less than or equal to the number permitted, then a
match is recorded.
[0255] The patterns and the DNA sequence are stored in Fasta-format
DNA sequence files. The length of the patterns and the DNA
sequences are limited only by available computer memory. The
computer program first loads the patterns into memory. Each DNA
sequence is then loaded sequentially from the Fasta file and
searched sequentially with each pattern, allowing for the mismatch
frequency designated by the user. The reverse complement of the DNA
sequence is generated and again searched with the patterns. The
coordinates of the pattern matches for each sequence and the name
of the pattern that matched are saved in memory. Once a sequence
has been searched with all patterns, the coordinates of the
patterns are sorted into order, and the name of the DNA sequence,
the name of the pattern, and the coordinates of the match are
written to an output file. TABLE-US-00003 LENGTHY TABLE REFERENCED
HERE US20070277267A1-20071129-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-00004 LENGTHY TABLE The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070277267A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070277267A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070277267A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References